B"NextPage" "quest19" "quest20" enterPage leavePage enterPage NextPage leavePage quest19 quest20 Suitable elution conditions for a carboxymethylcellulose column are:: Increasing salt concentration Decreasing salt concentration Increasing pH Decreasing pH Increasing ionic strengthhhhh In a gel filtration column: The smallest molecules emerge first The largest molecules emerge first There is a continuous gel matrix There is a stationary phase There is a mobile phase ase The largest molecules are always in the stationary phasen the stationary phase NextPage 4questscore19 4questscore20 default buttonUp buttonUp default questscore20 questscore19 quest19 quest20 Score Press the 'Score' button after finshing the questions to obtain your final score. 4questscore19 4questscore20 B"19a" B"19b" B"19c" B"19d" B"19e" B"20a" B"20b" B"20c" B"20d" B"20e" 4questscore18 4questscore17 4questscore16 4questscore15 4questscore14 4questscore13 4questscore12 4questscore11 4questscore10 4questscore9 4questscore8 4questscore7 4questscore6 4questscore5 4questscore4 4questscore3 --System count questscores --Obtain dialog box showing total no points "You scored" && out a possible 100" --shows nextpage B- avoids Bbeing pressed without --obtaining B"NextPage" default buttonUp buttonUp You scored points out of a possible 100 NextPage default count questscore1 questscore2 questscore3 questscore4 questscore5 questscore6 questscore7 questscore8 questscore9 questscore10 questscore11 questscore12 questscore13 questscore14 questscore15 questscore16 questscore17 questscore18 questscore20 questscore19 Basic 44(4(4 -d0&2v3k Answers to Techniques Questionss The answers to the problems that you got wrong can be seen below. Once you have read the correct answer click the mouse in the white box to move onto the next answer. NextPage default buttonUp buttonUp default answer20 "answer20" buttonUp buttonUp answer20 Question 20: In a gel filtration column, there is both a mobile and stationary phase- this leads to the largest molecules emerging first. The column is not a continuous gel, it consists of a number of separate gel beads. d CAP site respectively.to the DNA. answer19 "answer19" buttonUp buttonUp answer19 Question 19: Suitable elution conditions for a carboxymethylcellulose column are: 1. increasing the pH 2. increasing the salt concentration 3. increasing the ionic strength.ionnarged. of fibrinogen.. is the mass of the four fibrinopeptides. answer18 "answer18" buttonUp buttonUp answer18 Question 18: The initial conditions for a carboxymethylcellulose column are: 1. a pH of 5.0 2. a low salt concentration 3. the column should be negatively charged. f genomic light chain genes, and nuber of genomic heavy chain genes. answer17 "answer17" buttonUp buttonUp answer17 Question 17: 2-dimensional electrophoresis will separate molecules of the same size but differing pI; the same pI but differing size; and differing pI and size. cription.e and the e-amino group of lysine. answer16 "answer16" buttonUp buttonUp answer16 Question 16: DNA molecules of different sizes can be separated on both polyacrylamide gels and agarose gels. They all have the same charge/mass ratios and will migrate at the same rate in free solution. g" a hole in the foreign cell membrane. is achieved by platelet aggregation and requires calcium ions. answer15 "answer15" buttonUp buttonUp answer15 Question 15: During SDS PAGE, the SDS glycoprotein complexes have a more compact shape than the SDS polypeptide complexes, yet move at a slower rate due to their lower charge/mass ratio. embrane the phosphatidyl serine is incorporated into the outer surface of the plasma membrane. answer14 "answer14" buttonUp buttonUp answer14 Question 14: During SDS PAGE, all SDS polypeptide complexes migrate at a rate dependent on their charge/mass ratio. They may move at different rates through the gel pores. High concentrations of both factor Xa and prothrombin are generated on the aggregated platelet plasma membrane leading to a rate hundreds of times faster than in solution. answer13 "answer13" buttonUp buttonUp answer13 Question 13: In free solution, all SDS polypeptide complexes will migrate at the same rate- this rate is dependent on the charge/mass ratio. e endothelial cells and plasma. Platelets only come into contact with collagen when the blood vessel is damaged. answer12 "answer12" buttonUp buttonUp answer12 Question 12: During SDS PAGE, the formation of the rod shaped macromolecular complexes requires the presence of both sodium dodecyl sulphate and mercaptoethanol. factor which enhances the activity of factor Xa. Fibrinoligase is a transamidase - it breaks one amide bond to form another. answer11 "answer11" buttonUp buttonUp answer11 Question 11 Both sodium dodecyl sulphate and mercaptoethanol will cause unfolding of a polypeptide. It binds to the aggregated platelet surface via calcium ions. It is a proteolytic enzyme which converts prothrombin into thrombin.... answer10 "answer10" buttonUp buttonUp answer10 Question 10: Polymerisation of acrylamide and methylene bisacrylamide creates polyacrylamide. sicles have negatively charged phosopholipids on their inner surface so on fusing with the platelet plasma membrane the negative charges are transferred to the outer surface of the plasma membrane. polymerase or by forming a bend in the DNA.by forming a bend in the DNA. answer9 "answer9" buttonUp buttonUp answer9 Question 9: Mobility of SDS protein complexes is inversely proportional to the logMr. They are capable of differentiation and possess surface bound receptors.lymerase for the promoter by 100 fold. The result of this is that when lactose enters the cell the RNA polymerase is ready to start transcription immediately, leading to very quick production of the proteins. answer8 "answer8" buttonUp buttonUp answer8 Question 8: Mobility of DNA molecules is inversely proportional to the logMr. lexes to specific T cells. They then promote T cell maturation by releasing interleukins. They do not differentiate. r the allolactose concentration. enters the cell the RNA polymerase is ready to start transcription immediately, leading to very quick production of the proteins. answer7 "answer7" buttonUp buttonUp answer7 Question 7: In free solution, mobility of all DNA molecules is constant, as is mobility of SDS polypeptide complexes. antigen fragment complexes to T cells.red by platelet aggregation and the triggering of the coagulation system......eading to low lactose uptake into the cell. is made so only a small amount of lactose enters the cell. uction of the proteins. answer6 "answer6" buttonUp buttonUp answer6 Question 6: The molecules exposed to isoelectric focusing are exposed to both an electric field and a pH gradient. The molecules may move at different velocities depending on their charge and size. Their final position is determined by their isoelectric point. ount of lactose enters the cell. ion immediately, leading to very quick production of the proteins. answer5 "answer5" buttonUp buttonUp answer5 Question 5: 2-dimensional electrophoresis and SDS polyacrylamide gel electrophoresis both require molecules or complexes to have constant charge/mass ratios. have surface complementarity and are generally dissimilarly charged to the epitope.njury. only a small amount of lactose enters the cell. ion immediately, leading to very quick production of the proteins. answer4 "answer4" buttonUp buttonUp answer4 Question 4: 2-Dimensional electrophoresis separates molecules on the basis of both size and charge.chains. They form the bnding sites for epitopes and thus by definition, antigen binding sites tose because only a small amount of permease is made so only a small amount of lactose enters the cell. ion immediately, leading to very quick production of the proteins. answer3 "answer3" buttonUp buttonUp answer3 Question 3: Isoelectric focusing separates molecules on the basis of pI alone ut of these 4 chains, 2 are variable and 2 are constant. All 4 chains are linked by disuphide links.... ry of lactose because only a small amount of permease is made so only a small amount of lactose enters the cell. ion immediately, leading to very quick production of the proteins. answer2 "answer2" buttonUp buttonUp answer2 Question 2: Isoelectric focusing and ion exchange chromatography separate molecules on the basis of charge alone. es and are degraded by B lymphocytes (B cells) the enzyme can bind to the promoter. nly a small amount of permease is made so only a small amount of lactose enters the cell. ion immediately, leading to very quick production of the proteins. answer1 "answer1" buttonUp buttonUp answer1 Question 1: Gel filtration separates molecules on the basis of size alone. mphocytes. No platelets or erythrocytes are involved. hat control the entry and metabolism of lactose. ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? FirstPage buttonUp buttonUp 1st Page sysLockscreen ZhorizPos -300 "m1" &, 2670 "m1" 3525, 2670 H-300 "m2" n, 2985 "m2" 4245, 2985 "prot1" "prot2" )3150, 1470 "prot3" )3075, 1485 ZvertPos -300 "m3" 4245, "m3" 4245, 1920 "prot4" )3050, 1470 "prot5" )1815, 1830 "sds" "s1" 2250, 3225 "s13" 4703, 3967 "s4" 4148, 2107 "s9" 3083, 3202 "s5" 5250, 1245 "s2" 2453, 2782 "s6" 5558, 2542 "s10" 4140, 3000 "s8" 2670, 3915 "s7" 5558, 3622 "s3" 3525, 2070 "s12" 5273, 3307 "s11" 4995, 1890 "rod" default buttonUp buttonUp enterPage prot1 prot2 prot2 prot3 prot3 prot4 prot4 prot5 prot5 default vertPos:by vertPos:to vertPos horizPos:by horizPos:to horizPos BIBLIOGRAPHY Pharmacia- ION EXCHANGE CHROMATOGRAPHY Pages 4-7 7 - 109555555555555 backPage Previous default buttonUp buttonUp Previous default NextPage default buttonUp buttonUp default ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? FirstPage buttonUp buttonUp 1st Page Aims and objectives This is a self paced tutorial which aims to illustrate and explain some of the basic biochemical techniques commonly used to separate mixtures of macromolecules. The techniques included are: 1. Isoelectric focusing 2. SDS Polyacrylamide gel electrophoresis 3. DNA Electrophoresis 4. 2-Dimensional electrophoresis 5. Ion Exchange Chromatography 6. Gel filtration. There is also a concluding self test section which provides an opportunity to assess your understanding of the tutorial. sysLockscreen "paper" )90, 1455 "paper2" )90, 1455 "r1" )4140, 1500 "b1" )4140, 1500 "g1" )3600, 1530 "r1" 4240, 1500 "b1" 4165, 1500 "g1" 3500, 1530 0.25 "r1" 4340, 1500 "b1" 4190, 1500 "g1" 3400, 1530 0.25 "r1" 4440, 1500 "b1" 4215, 1500 "g1" 3300, 1530 0.25 "r1" 4540, 1500 "b1" 4240, 1500 "g1" 3200, 1530 0.25 "r1" 4640, 1500 "b1" 4265, 1500 "g1" 3100, 1530 0.25 "r1" 4740, 1500 "b1" 4290, 1500 "g1" 3000, 1530 0.25 "r1" 4840, 1500 "b1" 4315, 1500 "g1" 2900, 1530 0.25 "r1" 4940, 1500 "b1" 4340, 1500 "g1" 2800, 1530 0.25 "r1" 5040, 1500 "b1" 4365, 1500 "g1" 2700, 1530 0.25 "r1" 5140, 1500 "b1" 4390, 1500 "g1" 2600, 1530 0.25 "r1" 5240, 1500 "b1" 4415, 1500 "g1" 2500, 1530 0.25 "r1" 5340, 1500 "b1" 4440, 1500 "g1" 2400, 1530 0.25 "r1" 5440, 1500 "b1" 4465, 1500 "g1" 2300, 1530 "r1" 5540, 1500 "b1" 4490, 1500 "g1" 2200, 1530 0.25 "r1" 5640, 1500 "b1" 4515, 1500 "g1" 2100, 1530 0.25 "r1" 5740, 1500 "b1" 4540, 1500 "g1" 2000, 1530 0.25 "r1" 5840, 1500 "b1" 4565, 1500 "g1" 1900, 1530 0.25 "r1" 5940, 1500 "b1" 4590, 1500 "g1" 1800, 1530 0.25 "r1" 6040, 1500 "b1" 4615, 1500 "g1" 1700, 1530 0.25 "r1" 6140, 1500 "b1" 4640, 1500 "g1" 1600, 1530 0.25 "r1" 6240, 1500 "b1" 4665, 1500 "g1" 1500, 1530 0.25 "r1" 6340, 1500 "b1" 4690, 1500 "g1" 1400, 1530 0.25 "r1" 6440, 1500 "b1" 4715, 1500 "g1" 1300, 1530 0.25 "r1" 6540, 1500 "b1" 4740, 1500 "g1" 1200, 1530 0.25 "r1" 6640, 1500 "b1" 4765, 1500 "g1" 1100, 1530 0.25 "r1" 6740, 1500 "b1" 4790, 1500 "g1" 1005, 1530 0.25 "r1" 6840, 1500 "b1" 4790, 1500 "g1" "g2" )990, 1530 0.25 "r1" 6915, 1500 "b1" 4815, 1500 "g2" "g3" )1005, 1530 0.25 "r1" "r2" )7035, 1515 "b1" 4840, 1500 0.25 "r2" "r3" )7125, 1515 "b1" 4865, 1500 0.25 "b1" 4890, 1500 0.25 "b1" 4915, 1500 0.25 "b1" 4940, 1500 0.25 "b1" 4965, 1500 0.25 "b1" 4990, 1500 0.25 "b1" 5015, 1500 0.25 "b1" 5040, 1500 0.25 "b1" 5065, 1500 0.25 "b1" 5090, 1500 0.25 "b1" 5115, 1500 0.25 "b1" 5140, 1500 0.25 "b1" 5165, 1500 0.25 "b1" 5250, 1500 0.25 "b1" "b2" )5370, 1515 0.25 "b2" "b3" )5460, 1515 0.25 default buttonUp buttonUp paper paper paper2 default sysLockscreen "block" "Glyco" 1780, 1575 "polypep" 1865, 2385 1880, 1575 2065, 2385 1980, 1575 2265, 2385 2080, 1575 2465, 2385 2180, 1575 2665, 2385 2280, 1575 2865, 2385 2380, 1575 3065, 2385 2480, 1575 3265, 2385 2580, 1575 3465, 2385 2680, 1575 3665, 2385 2780, 1575 3865, 2385 2880, 1575 4065, 2385 2980, 1575 4265, 2385 3080, 1575 4465, 2385 3180, 1575 4665, 2385 3280, 1575 4865, 2385 3380, 1575 5065, 2385 3480, 1575 5265, 2385 3580, 1575 5465, 2385 3680, 1575 5665, 2385 3780, 1575 5865, 2385 3880, 1575 6065, 2385 3980, 1575 6150, 2385 )735, 2085 default buttonUp buttonUp enterPage block Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep Glyco polypep block default Uv4x4x4 sysLockscreen "bloc2" "bloc1" )420, 1785 B"animate" "t1" )870, 3120 "title" "isogel" )420, 1695 "isogel2" "sdsgela" "sdsgel3" U"fr" U"fg" U"fb" U"fv" U"ff" enterPage leavePage enterPage bloc2 bloc1 animate title isogel isogel2 sdsgela sdsgel3 leavePage #D#t# $4$H' -$.T. /D/t/ "sdsgel" nthe 1140, 1365 5340, 2385 5340, 2385 buttonUp buttonUp sdsgel The proteins are thus separated on the basis of both size and charge. dimensions. sdsgela polyacrylamide gel sdsgel3 polyacrylamide gel isogel2 buttonUp buttonUp isoelectric focusing gel roddraperfocussing paper The gel is then infused with SDS and mercaptoethanol,.e. nthe 870, 3120 870, 3120 855, 3120 855, 3120 buttonUp buttonUp and then joined to a slab of polyacrylamide gel......e. title 2-DIMENSIONAL ELECTROPHORESISSS -A POWERFUL SEPARATION TECHNIQUEE IN POLYACRYLAMIDE GEL : animate Animate isogel buttonUp buttonUp isoelectric focusing gel roddraperfocussing paper 2-D electrophoresis is a useful technique for separation of proteins on the basis of both charge and size. The first stage of this is to perform isoelectric focusing on the mixture of proteins in a polyacrylamide rod gel.....d.el rod. The molecules are thus separated on the basis of charge. nthe 4215, 1740 4455, 1755 4545, 1755 4455, 1755 buttonUp buttonUp U"fr" nthe 6900, 1740 U"fg" 5835, 1740 U"fb" 2460, 1740 buttonUp buttonUp nthe 2040, 2130 2025, 2475 2024, 3210 2025, 3780 2025, 4065 3885, 1965 3885, 2250 3885, 2700 3885, 3420 4545, 2130 4545, 3090 4560, 3870 buttonUp buttonUp nthe 2025, 2700 2025, 2430 2025, 2205 2040, 1980 3915, 2460 3900, 2220 3885, 1995 4545, 2235 4545, 2010 buttonUp buttonUp nthe 1425, 1755 3315, 1755 )1890, 2205 U"ff" 1425, 1755 U"fv" 3315, 1755 buttonUp buttonUp bloc1 nthe 420, 1785 buttonUp buttonUp bloc2 nthe 5340, 1380 5325, 1380 buttonUp buttonUp SDS-polyacrylamide gel electrophoresis is then performed in the second dimension ..... "sdsgel" nthe 1140, 1365 5340, 2385 5340, 2385 buttonUp buttonUp sdsgel REPEAT OR MOVE TO NEXT PAGEEE..........basis of both size and charge. dimensions. sysLockscreen "block" )870, 2265 "elec" )870, 2280 "title" "gel" )1358, 1545 "big" )1665, 2805 )1665, 2280 "small" )1665, 1785 B"animate" enterPage leavePage enterPage block title small animate leavePage &L&z& &h(^*R+ nthe 1358, 1545 buttonUp buttonUp animate Animate The situation is similar for movement of DNA and nucleic acids. However, the high phosphate content of nucleic acids imparts the same charge/mass ratio for all molecules of this type. Thus, nucleic acids are separated in polyacrylamide gels on the basis of their size. Agarose is also commonly used as the medium in separation gels..... ` ^)1 block nthe 840, 2310 870, 2265 title DNA ELECTROPHORESISSSSSSSSSSSSSS USING POLYACRYLAMIDE OR AGAROSE :::::::::::::::::::::::::::::: o &54 small title )570, 285 sysLockscreen enterPage leavePage enterPage title leavePage title Ion exchange chromatography is a technique which allows separation proteins on the basis of their charge differences. An ion exchange column is used which consists of an insoluble matrix carrying either positively or negatively charged groups. A mixture of proteins is added to the column under conditions where most carry a charge opposite to that of the column and most proteins bind electrostatically to the column. A carboxymethyl column is negatively charged and to render most proteins positively charged the pH has to be around 5.0. A lower pH will suppress the ionization of the carboxymethyl groups. For a positively charged DEAE column the pH has to be around 8 where most proteins are negatively charged. In both case the ionic strength has to be low - usually less than 0.01. This is because high concentration of ions of opposite charge to the column matrix will reduce the affinity of the proteins for the column...n exchange chromatography is therefore a very high resolution separation technique...........ctive separation technique.tive separation technique. title ION EXCHANGE CHROMATOGRAPHYYYSS - INITIAL CONDITIONSEEEEEEEEEEEEE IN POLYACRYLAMIDE GEL : sysLockscreen "erty" 4515, 1755, 7500, 4110 0.25 4545, 1755, 7500, 4110 0.25 4590, 1755, 7500, 4110 0.25 4620, 1755, 7500, 4110 0.25 4650, 1755, 7500, 4110 0.25 4665, 1755, 7500, 4110 0.25 4710, 1755, 7500, 4110 0.25 4755, 1755, 7500, 4110 0.25 4800, 1755, 7500, 4110 0.25 4830, 1755, 7500, 4110 0.25 4845, 1755, 7500, 4110 0.25 4875, 1755, 7500, 4110 0.25 4920, 1755, 7500, 4110 0.25 4950, 1755, 7500, 4110 ZvertPos 1845 3210 "rband" 3390, 3390, 3210 "rtip" )3390,3330 "beek" "rbeek" )3470, 3825 )3470, 3825 0.25 5130, 1755, 7500, 4110 0.25 5160, 1755, 7500, 4110 0.25 5205, 1755, 7500, 4110 0.25 5250, 1755, 7500, 4110 0.25 5295, 1755, 7500, 4110 0.25 5340, 1755, 7500, 4110 0.25 5355, 1755, 7500, 4110 0.25 5370, 1755, 7500, 4110 0.25 5400, 1755, 7500, 4110 0.25 5445, 1755, 7500, 4110 0.25 5460, 1755, 7500, 4110 0.25 5475, 1755, 7500, 4110 0.25 5505, 1755, 7500, 4110 0.25 5520, 1755, 7500, 4110 0.25 5550, 1755, 7500, 4110 0.25 5595, 1755, 7500, 4110 0.25 5625, 1755, 7500, 4110 0.25 5655, 1755, 7500, 4110 0.25 5790, 1755, 7500, 4110 0.25 5820, 1755, 7500, 4110 0.25 6000, 1755, 7500, 4110 0.25 "mixband" 1845 3210 "gband" 3390, 3390, 3210 "gtip" )3390,3330 "gbeek" )3470, 3825 )3470, 3825 0.25 6030, 1755, 7500, 4110 0.25 6060, 1755, 7500, 4110 0.25 6105, 1755, 7500, 4110 0.25 6195, 1755, 7500, 4110 0.25 6240, 1755, 7500, 4110 0.25 6300, 1755, 7500, 4110 0.25 6345, 1755, 7500, 4110 0.25 6390, 1755, 7500, 4110 0.25 6420, 1755, 7500, 4110 0.25 6465, 1755, 7500, 4110 0.25 6510, 1755, 7500, 4110 0.25 6540, 1755, 7500, 4110 0.25 6570, 1755, 7500, 4110 0.25 6645, 1755, 7500, 4110 0.25 6720, 1755, 7500, 4110 0.25 6780, 1755, 7500, 4110 0.25 6825, 1755, 7500, 4110 0.25 0.25 7095, 1755, 7500, 4110 1845 3210 "pband" 3390, 3390, 3210 "ptip" )3390,3330 "pbeek" )3470, 3825 )3470, 3825 0.25 6960, 1755, 7500, 4110 0.25 7005, 1755, 7500, 4110 0.25 7050, 1755, 7500, 4110 0.25 7170, 1755, 7500, 4110 0.25 7230, 1755, 7500, 4110 0.25 7275, 1755, 7500, 4110 0.25 7335, 1755, 7500, 4110 0.25 7380, 1755, 7500, 4110 0.25 7440, 1755, 7500, 4110 0.25 7500, 1755, 7515, 4110 default buttonUp buttonUp enterPage rband rband rband rbeek rbeek mixband gband gband gband gbeek gbeek pband pband pband pbeek pbeek default vertPos:by vertPos:to vertPos "t1" )315, 1650 B"test" "erty" 4485, 1755, 7500, 4110 )4500, 1755 "graf" )4485, 1740 title )570, 285 "beek" )3470, 3825 "column" )3380, 1680 "mixband" )3390, 1845 "rband" )3390, 1845 "pband" )3390, 1845 "gband" )3390, 1845 "rtip" "gtip" "ptip" "rbeek" "gbeek" "pbeek" sysLockscreen enterPage leavePage enterPage column mixband rband pband gband rbeek gbeek pbeek title leavePage Protein conc'n conc'n conc'nn'n Increasing Fraction Number title ION EXCHANGE CHROMATOGRAPHYYSSS - AT THE MACROSCOPIC LEVELVELYEEE IN POLYACRYLAMIDE GEL : Animate rbeek 3915, 4920 3660, 4290 "rbeek" nthe 3470, 3825 buttonUp buttonUp rbeek nthe 315, 1650 buttonUp buttonUp The graph (right) shows the elution of the three different proteins as the concentration of NaCl increases. The protein eluted first has lowest charge while those eluted later have higher charges....fractions.ns aswell as the protein concentrations in the same fractions. gbeek pbeek column "column" nthe 2880, 1680 buttonUp buttonUp column "rtip" nthe 3390, 3330 buttonUp buttonUp pband "pband" nthe 3390, 2640 3375, 1845 3390, 3210 rband gband mixband of selection to 750 title )570, 285 sysLockscreen enterPage leavePage enterPage title leavePage title title ION EXCHANGE CHROMATOGRAPHYYYSS - PROTEIN ELUTIONONSEEEEEEEEEEEEE IN POLYACRYLAMIDE GEL : Elution of the bound proteins is achieved using increasing concentrations of competing ions, usually Na or Cl depending on the charge of the column matrix. The more highly charged the bound macromolecule, the greater affinity it has for the column. This means that the the more highly charged proteins require a higher concentration of competing ions for their elution. By increasing the ionic strength of the elution buffer it is possible to separate proteins which differ by just one charge unit. Ion exchange chromatography is therefore a very high resolution separation technique...l the stuff about it taking many Cl- to elute the differently charged proteins, and the next page provides a diagramatic version only, and the proteins are much more charged in reality. "quest5" "quest6" leavePage leavePage quest5 quest6 Which separation technique(s) require molecules or complexes to have constant charge/mass ratios?atios? Gel filtration Isoelectric focusing 2-Dimensional electrophoresis Ion exchange chromatography SDS polyacrylamide electrophoresiss The molecules subjected to isoelectric focusing in polyacrylamide are : Exposed to a pH gradient Exposed to an electric field Exposed to a gradient gel Move at a constant velocity Move to a position determined by their mass larity?????? quest5 quest6 NextPage 4questscore5 4questscore6 B"5a" B"5b" B"5c" B"5d" B"5e" B"6a" B"6b" B"6c" B"6d" B"6e" default buttonUp buttonUp default questscore6 questscore5 "quest7" "quest8" leavePage leavePage quest7 quest8 When subjected to electrophoresis in free solution:polypeptide complexes: Mobility of DNA molecules is inversely proportional to the M Mobility of SDS polypeptide complexes is proportional to the M Mobility of DNA molecules is inversely proportional to the logM Mobility of SDS polypeptide complexes is proportional to the logM Mobility of all DNA molecules is constantMMMMMMMMMMMMMMMMMMMMMMMrrrrrrMrMrrrrheir Mr When subjected to electrophoresis in polyacrylamide gel: Mobility of DNA molecules is inversely proportional to the M Mobility of DNA molecules is proportional to the M Mobility of DNA molecules is inversely proportional to the logM Mobility of DNA molecules is proportional to the logM Mobility of DNA molecules is not related to the M o their logM t related to their logM quest7 quest8 NextPage 4questscore7 4questscore8 B"7a" B"7b" B"7c" B"7d" B"7e" B"8a" B"8b" B"8c" B"8d" B"8e" default buttonUp buttonUp default questscore8 questscore7 mediately, lea sysLockscreen ZvertPos -2000 "p2" 4175, "p2" 4175, 890 nthe 4070, 1070 3950, 1130 3800, 1145 3680, 1145 -2000 "p3" 4175, "p3" 4175, 890 4250, 965 4400, 1070 4475, 1160 4565, 1205 4625, 1235 4715, 1355 -2000 "p4" 4175, "p4" 4175, 890 4190, 1040 4190, 1250 4175, 1445 4160, 1595 4130, 1805 4025, 1820 3920, 1850 3815, 1865 3665, 1865 )3990, 1215 "2rep" )3665, 1160 "p2" 3830, 1295 )4560, 1605 "p2" 3980, 1460 )4155, 2025 "p2" 4130, 1715 )4050, 2715 "p2" 4205, 2030 )4560, 3105 "p2" 4280, 2360 )4020, 3540 "p2" 4295, 2930 )4245, 4140 "p2" 4295, 3245 )4515, 4650 "p2" 4325, 3620 )4335, 5535 "p2" 4340, 3950 "p2" 4355, 4655 "p2" 4385, 5375 "10" )4410, 1185 )4835, 1400 "p3" 4430, 1715 "11" )4050, 1740 "12" )4575, 2115 "p3" 4280, 1955 "13" )3975, 2325 "14" )4485, 2550 "p3" 4235, 2195 "15" )4005, 3105 "16" )4365, 3540 "p3" 4220, 2600 "17" )3840, 4065 "18" )4050, 4455 "p3" 4220, 3185 "19" )4815, 4575 "20" )3990, 4740 "p3" 4190, 3905 "21" )4410, 5010 "22" )3945, 5145 "p3" 4145, 4760 "23" )4845, 5505 "24" )3870, 5640 "25" )4815, 1140 "26" )4215, 1485 "27" )3915, 1455 )3680, 1910 "28" )4455, 1890 "43" )4365, 2310 "p4" 3785, 1985 "29" )4845, 2520 "30" )4290, 2865 "31" )3765, 3645 "32" )4215, 3270 "p4" 4010, 2570 "33" )4485, 3780 "34" )4050, 3870 "35" )4560, 4095 "36" )3765, 4530 "37" )4365, 4410 "p4" 4025, 3035 "38" )4260, 4785 "39" )4710, 5025 "p4" 4145, 4085 "40" )4260, 5265 "41" )4005, 5385 "p4" 4130, 5315 "42" )4560, 5295 default buttonUp buttonUp enterpage default vertPos:by vertPos:to vertPos "quest9" "quest10" leavePage leavePage quest9 quest10 Polymerisation of which two molecules creates polyacrylamide? Persulphate Acrylamide Ethylene bisacrylamide Bismethylene acrylamide Methylene bisacrylamidee quest9 quest10 NextPage 4questscore9 4questscore10 B"9a" B"9b" B"9c" B"9d" B"9e" B"10a" B"10b" B"10c" B"10d" B"10e" default buttonUp buttonUp default questscore10 questscore9 Mobility of SDS-protein complexes is inversely proportional to theM Mobility of SDS-protein complexes is proportional to the M Mobility of SDS-protein complexes is inversely proportional to the logM Mobility of SDS-protein complexes is proportional to the logM Mobility of SDS-protein complexes is not related to the M ersely proportional to their M Mobility of DNA molecules is proportional to their M Mobility of DNA molecules is inversely proportional to their logM Mobility of DNA molecules is proportional to their logM Mobility of DNA molecules is not related to their M to their M When subjected to electrophoresis in polyacrylamide gel: "quest11" "quest12" leavePage leavePage quest11 quest12 Which of the following will cause unfolding of a polypeptide? Methylene bisacrylamide Sodium dodecyl sulphate !% mercaptoethanol Dimercaptoethanol Persulphate ions During SDS-PAGE the formation of the rod-shaped macromolecular complexes:::::::::?tide stabilised? Requires the presence of SDS Requires the presence of polyacrylamide Is induced by the electric field Requires the presence of mercaptoethanol Requires the presence of persulphate ionsof persulphateatelarge to diffuse into the gel beads beads quest11 quest12 NextPage 4questscore11 4questscore12 B"11a" B"11b" B"11c" B"11d" B"11e" B"12a" B"12b" B"12c" B"12d" B"12e" default buttonUp buttonUp default questscore12 questscore11 sysLockscreen B"animate" "aps" )2880, 1305 "ac1" )1725, 2790 "ac2" )3555, 2790 "ac3" )5460, 2820 "title" sysLocscreen enterPage leavePage enterPage animate title sysLocscreen leavePage sysLocscreen ~#<(r) 506r<`C Persulphate initiator Forms 2 radicalsSRRRR Acrylamide moleculesSS A sulphate radical initiates polymerisation of acrylamideEEEEEEEw "aps" nthe 2880, 1305 buttonUp buttonUp apsrad2 nthe 4095, 1305 buttonUp buttonUp n!F!k! apsrad1 title SDS-PAGE ELECTROPHORESISS b):)_) GEL FORMATION (2) animate Animate "sul" nthe 570, 2700 buttonUp buttonUp p,H,m, \-4-Y- "ac6" nthe 8355, 2835 "ac5" 7455, 2835 NH 22 HH 22 HH 22 HH 22 Chain propagation continues: NH 22 HH 22 HH 22 HH 22 blcl HH 22 HH 22 Jn@nGn polac2 NH 22 HH 22 Xs0sUs HH 22 HH 22 "polac1" nthe 2580, 2490 buttonUp buttonUp polac1 "acrad2" nthe 3510, 2520 3510, 2505 buttonUp buttonUp acrad2 Hy yEy NH 22 HH 22 HH 22 p|H|m| HH 22 nthe 2580, 2505 buttonUp buttonUp NH 22 HH 22 HH 22 HH 22 "ac3" nthe 5460, 2820 buttonUp buttonUp NH222 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 polac4 "acrad2" nthe 3510, 2520 3510, 2505 buttonUp buttonUp acrad2 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 nthe 2580, 2505 buttonUp buttonUp NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 "ac5" nthe 7455, 2835 buttonUp buttonUp NH222 HH 22 HH 22 HH 22 polac5 nthe 3585, 2505 3600, 2490 buttonUp buttonUp "acrad3" nthe 5415, 2520 buttonUp buttonUp acrad3 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 nthe 2580, 2505 buttonUp buttonUp NH 22 HH 22 HH 22 HH 22 "quest13" "quest14" leavePage leavePage quest13 quest14 In free solution, all SDS-polypeptide complexes: Will migrate at the same rate Migrate at a rate dependent on mass Migrate at a rate dependent on charge Migrate at a rate dependent on charge/mass ratio May migrate at different ratesss In polyacrylamide gel electrophoresis, all SDS-polypeptide complexes: Will migrate at the same rate Migrate at a rate dependent on mass Migrate at a rate dependent on charge Migrate at a rate dependent on charge/mass ratio May migrate at different ratesss NextPage 4questscore13 4questscore14 B"13a" B"13b" B"13c" B"13d" B"13e" B"14a" B"14b" B"14c" B"14d" B"14e" default buttonUp buttonUp default questscore14 questscore13 quest13 quest14 "quest15" "quest16" leavePage leavePage quest15 quest16 During SDS-PAGE the SDS-glycoprotein complexes::uivalent mass:sof equivalent mass::::::::::::::::: Move faster than complexes of SDS and the equivalent mass polypeptide Move slower than complexes of SDS and the equivalent mass polypeptide Have a more compact shape than SDS-polypeptide complexes Have a lower charge/mass ratio than SDS-polypeptide complexes Have a higher charge/mass ratio than SDS-polypeptide complexes d like" DNA molecules of different sizes: Can be separated by electrophoresis in a agarose gel Can be separated by electrophoresis in a polyacrylamide gel All have differing charge/mass ratios Require the addition of SDS prior to separation Will migrate at the same rate in free solutionionnnnnnnn NextPage 4questscore15 4questscore16 B"15a" B"15b" B"15c" B"15d" B"15e" B"16a" B"16b" B"16c" B"16d" B"16e" default buttonUp buttonUp default questscore16 questscore15 quest15 quest16 "quest17" "quest18" leavePage leavePage quest17 quest18 2-Dimensional electrophoresis::ill separate molecule mixtures: Will separate polypeptides of the same size and differing pI Will separate polypeptides of the same pI and differing size Will separate polypeptides of the same pI and size Will separate polypeptides of differing pI and size Uses SDS-PAGE in the first dimension The initial conditions for a carboxymethylcellulose column are: A pH of 8.0 A pH of 5.0 A low salt concentration A high salt concentration The column is negatively charged NextPage 4questscore17 4questscore18 B"17a" B"17b" B"17c" B"17d" B"17e" B"18a" B"18b" B"18c" B"18d" B"18e" default buttonUp buttonUp default questscore18 questscore17 quest17 quest18 sysLockscreen "matrix2" B"animate" "title" B"FirstPage" B"ExitProgram" B"backPage" B"NextPage" "p2" )4165, -2000 "p3" )4270, -3000 "p4" )3985, -2000 "2rep" enterPage leavePage enterPage matrix2 animate title FirstPage ExitProgram backPage NextPage leavePage "erty" 4515, 1755, 7500, 4110 4545, 1755, 7500, 4110 4590, 1755, 7500, 4110 4620, 1755, 7500, 4110 4650, 1755, 7500, 4110 4665, 1755, 7500, 4110 4710, 1755, 7500, 4110 4755, 1755, 7500, 4110 4800, 1755, 7500, 4110 4830, 1755, 7500, 4110 4845, 1755, 7500, 4110 4875, 1755, 7500, 4110 4920, 1755, 7500, 4110 4950, 1755, 7500, 4110 4980, 1755, 7500, 4110 5040, 1755, 7500, 4110 5085, 1755, 7500, 4110 5130, 1755, 7500, 4110 5160, 1755, 7500, 4110 5205, 1755, 7500, 4110 5250, 1755, 7500, 4110 5295, 1755, 7500, 4110 5340, 1755, 7500, 4110 5355, 1755, 7500, 4110 5370, 1755, 7500, 4110 5400, 1755, 7500, 4110 5445, 1755, 7500, 4110 5460, 1755, 7500, 4110 5475, 1755, 7500, 4110 5505, 1755, 7500, 4110 5520, 1755, 7500, 4110 5550, 1755, 7500, 4110 5595, 1755, 7500, 4110 5625, 1755, 7500, 4110 5655, 1755, 7500, 4110 5790, 1755, 7500, 4110 5820, 1755, 7500, 4110 5850, 1755, 7500, 4110 5880, 1755, 7500, 4110 5925, 1755, 7500, 4110 5970, 1755, 7500, 4110 6000, 1755, 7500, 4110 6030, 1755, 7500, 4110 6060, 1755, 7500, 4110 6105, 1755, 7500, 4110 6195, 1755, 7500, 4110 6240, 1755, 7500, 4110 6300, 1755, 7500, 4110 6345, 1755, 7500, 4110 6390, 1755, 7500, 4110 6420, 1755, 7500, 4110 6465, 1755, 7500, 4110 6510, 1755, 7500, 4110 6540, 1755, 7500, 4110 6570, 1755, 7500, 4110 6645, 1755, 7500, 4110 6720, 1755, 7500, 4110 6780, 1755, 7500, 4110 6825, 1755, 7500, 4110 6855, 1755, 7500, 4110 6900, 1755, 7500, 4110 6960, 1755, 7500, 4110 7005, 1755, 7500, 4110 7050, 1755, 7500, 4110 7095, 1755, 7500, 4110 7170, 1755, 7500, 4110 7230, 1755, 7500, 4110 7275, 1755, 7500, 4110 7335, 1755, 7500, 4110 7380, 1755, 7500, 4110 7440, 1755, 7500, 4110 7500, 1755, 7515, 4110 buttonUp buttonUp --removes cross the answer boxes "quest1" "quest2" leavePage leavePage quest1 quest2 Which technique(s) separate on the basis of size alone???????????????????????????????? Gel filtration Isoelectric focusing 2-Dimensional electrophoresis Ion exchange chromatography SDS polyacrylamide electrophoresishoresis Which technique(s) separate on the basis of charge alone? Gel filtration Isoelectric focusing 2-Dimensional electrophoresis Ion exchange chromatography SDS polyacrylamide electrophoresis 1. quest1 quest2 NextPage --see --questscore1 question 1. It zero Fincreases decreases depending on which answer boxes are --same applies questscores 2-20 --All forthcoming fquestions on have Jlayout except 4questscore2 B"1a" B"1b" B"1c" B"1d" B"1e" B"2a" B"2b" B"2c" B"2d" B"2e" default buttonUp buttonUp default questscore2 questscore1 4drop < 42 < + 1) sysLockscreen terPage enterPage leavePage enterPage leavePage F t .!^! "N"~" #>#n# GEL FILTRATION (1)))))))) A gel filtration column consists of two fluid phases - that within the gel beads which is stationary and that between the gel beads which is mobile. Macromolecules in the mobile phase may have no access, limited access or complete access to the stationary phase depending upon their size. Large molecules pass straight down the column in the mobile phase (right). Molecules with access to the stationary phase shuttle between the two phases. Their mobility down the column is inversely related to the proportion of the stationary phase available to them. The smallest molecules have complete access to the mobile phase and move slowest. Molecules with more limited access move at and intermediate velocity.sized macromolecules will spend experience both phases. e both phases. The gel beads consist of a three dimensional mesh with pore sizes of the same order of magnitude as the macromolecules to be separated. 4drop < 12 < + 1) sysLockscreen terPage enterPage leavePage enterPage leavePage bacteriaah2 ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? THE END FirstPage buttonUp buttonUp 1st Page Questions FirstPage 1st Page This section contains 20 questions based on the information in the previous pages which will take about 10 minutes to complete. Once in the quiz it is only possible to go forwards - it is not possible to exit or return to previous pages. N.B. For each question there are 5 checkboxes. The correct answer may involve the checking of any number of boxes from 0 to 5. GOOD LUCK! backPage Previous default buttonUp buttonUp Previous default NextPage default buttonUp buttonUp default ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? buttonUp buttonUp sysLockscreen "gel" "big" "lit" "inv" "permtext" B"test" "title" "rep" enterPage leavePage enterPage permtext title leavePage Stat. Mobile title GEL FILTRATION (2)))))))) nthe 1130, 2330 1590, 4430 1620, 4445 1635, 4475 1650, 4490 1665, 4520 1680, 4535 1695, 4550 1710, 4565 2310, 4105 2325, 4105 1575, 3795 2310, 3485 2325, 3485 ZhorizPos ZvertPos 2325 1575 2325, 2865 1590, 2555 1575, 3175 1575, 2555 2310, 2245 2325, 2245 1575, 1935 2305, 1625 2320, 1625 1595, 1465 1610, 1465 1595, 1465 975, 4350 1920, 1185 1920, 4365 "lit" nthe 1175, 1940 1190, 1940 2370, 3045 Animate permtext The diagram on the left represents both phases of a single gel filtration column. A macromolecule may spend time in either or both phases during filtration. The animation demonstrates the separation action of gel filtration on a mixture of three different sized macromolecules:::: The largest macromolecules are too wide to fit into the gel bead pores (whilst inside the pores, a macromolecule is in the stationary phase). This means that they are mobile at all times and will take the least time to pass through the gel filtration column. Therefore the fractions eluted first from the filtration will contain the largest macromolecules from the mix. The macromolecules of intermediate size will fit into the larger gel bead pores, and during this time will be in the stationary phase . In time, the molecules will move out of the various large pores and progress through the column. In this case, the time spent between mobile phases causes a slower rate of elution. ie, the intermediate sized molecules are eluted in the middle fractions.ules. nthe 2580, 2740 3090, 2895 The smallest macromolecules will fit into most of the gel bead pores and will thus experience more time in the stationary phase than the intermediate sized molecules. The effect of this is that the smallest molecules will take the longest time to pass through the column, and thus will be eluted in the final fractions. "rep" )3300, 3165 buttonUp buttonUp REPEAT OR MOVE TO THE NEXT PAGE sysLockscreen Send nthe 2625, 3300 2610, 3480 "117" 2505, 3630 "113" 2370, 3165 2190, 3180 "116" 2010, 3225 "114" 1830, 3285 "137" 2400, 3780 "135" 2280, 3915 2160, 4050 "73a" )1965, 4185 "112" 1650, 3345 "112a" )1365, 3465 1095, 1995 930, 2025 750, 2070 570, 2115 "56a" )315, 2220 1425, 2055 1575, 2160 1695, 2280 1830, 2400 1950, 2520 2100, 2595 "101" 2235, 2715 "109" 2385, 2790 2490, 2895 "108" 2595, 2955 "136" 2835, 3120 "134" 3000, 3180 "118" 3180, 3210 3345, 3225 3510, 3225 "138" 3675, 3210 "139" 3855, 3180 "133" 4035, 3165 4215, 3150 "141" 4395, 3135 "119" 4575, 3105 "120" 4740, 3075 "104" 4920, 3045 5055, 2985 "121" 5235, 2970 5400, 2925 "123" 5550, 2880 "147" 5760, 2820 "123" 5580, 2880 6090, 2655 "148" 6255, 2595 6420, 2520 "146" 6570, 2445 6720, 2370 "145" 6870, 2280 "143" 6990, 2190 7110, 2070 "124" 6015, 3030 "106" 6225, 2865 6390, 2925 6540, 3000 "105" 5880, 3150 5730, 3270 5385, 3750 5400, 3735 5745, 3255 5595, 3345 "103" 5415, 3345 5250, 3270 5085, 3180 4995, 3090 4890, 2880 4875, 2700 4860, 2520 4845, 2340 4830, 2160 4845, 1980 4905, 1815 4995, 1665 5100, 1530 5250, 1425 5415, 1395 5565, 1500 5685, 1620 5775, 1710 6075, 1740 6240, 1650 6375, 1545 6495, 1440 6570, 1290 "19a" )6570, 1050 7230, 1965 "40a" )7365, 1770 5940, 2040 6120, 2100 6300, 2145 6480, 2190 6645, 2250 5610, 1935 1200, 1710 1050, 1725 1230, 1740 900, 1650 "20a" )660, 1575 1560, 1845 1545, 1830 1710, 1875 1890, 1890 2070, 1875 2235, 1860 2400, 1815 2820, 1740 2460, 1815 2280, 1860 2100, 1890 1920, 1890 1740, 1890 1560, 1860 1290, 1845 3000, 1725 3180, 1710 3345, 1695 3540, 1695 3705, 1725 3525, 1710 3885, 1755 4065, 1770 "102" 4245, 1800 4410, 1830 4605, 1860 3420, 1425 4875, 1845 4770, 1875 5055, 1875 5235, 1890 5430, 1905 5250, 1890 2490, 1485 2310, 1455 2115, 1350 1965, 1200 "24a" )1785, 1155 2160, 1365 2040, 1260 2805, 1500 2970, 1425 3135, 1335 3300, 1275 3465, 1245 3645, 1275 "110" 3765, 1395 660, 3705 3090, 2970 "111" 3885, 1530 3960, 1680 "128" 3990, 1875 "129" 3915, 2025 "140" 3810, 2160 "130" 3735, 2310 "142" 3645, 2460 3555, 2610 "132" 3510, 2790 "131" 3480, 2970 "144" 3480, 3120 "149" 3465, 3330 "125" 3480, 3495 "150" 3525, 3660 "151" 3585, 3825 "126" 3645, 3990 "126a" )3750, 4125 6690, 3075 "107" 6825, 3180 "127" 6960, 3285 "127a" )7065, 3390 6855, 2475 "100" 7020, 2520 7200, 2595 7350, 2640 "68a" )7470, 2745 default buttonUp 'buttonUp enterPage default sysLockscreen "block" "big" 1765,2805 1815, 2280 "small" 1865, 1785 1865,2805 1965, 2280 2065, 1785 1965,2805 2115, 2280 2265, 1785 2065,2805 2265, 2280 2465, 1785 2165,2805 2415, 2280 2665, 1785 2265,2805 2565, 2280 2865, 1785 2365,2805 2715, 2280 3065, 1785 2465,2805 2865, 2280 3265, 1785 2565,2805 3015, 2280 3465, 1785 2665,2805 3165, 2280 3665, 1785 2765,2805 3315, 2280 3865, 1785 2865,2805 3465, 2280 4065, 1785 2965,2805 3615, 2280 4265, 1785 3065,2805 3765, 2280 4465, 1785 3165,2805 3915, 2280 4665, 1785 3265,2805 4065, 2280 4865, 1785 3365,2805 4215, 2280 5065, 1785 3465,2805 4365, 2280 5265, 1785 3565,2805 4515, 2280 5465, 1785 3665,2805 4665, 2280 5665, 1785 3765,2805 4815, 2280 5865, 1785 3865,2805 4965, 2280 6065, 1785 3965,2805 5115, 2280 6265, 1785 4065,2805 5265, 2280 6465, 1785 4165,2805 5415, 2280 6665, 1785 4265,2805 5565, 2280 6865, 1785 4365,2805 5715, 2280 6945, 1785 )870, 2265 "graph" default buttonUp buttonUp enterPage block small small small small small small small small small small small small small small small small small small small small small small small small small small small block graph default sysLockscreen "t1" )2580, 2740 "big" )1430, 1190 movement nthe 1430, 1190 1400, 1205 1385, 1235 1370, 1250 1340, 1295 1370, 1295 1400, 1340 1430, 1370 1475, 1415 1505, 1430 1460, 1475 1415, 1520 1370, 1550 1355, 1580 1385, 1595 1400, 1625 1445, 1640 1475, 1670 1490, 1700 1460, 1745 1415, 1760 1400, 1775 1385, 1805 1370, 1820 1355, 1835 1385, 1850 1430, 1880 1460, 1910 1475, 1940 1505, 1970 1475, 1985 1445, 2030 1415, 2045 1385, 2090 1370, 2120 1355, 2150 1340, 2180 1370, 2195 1400, 2225 1460, 2255 1490, 2285 1505, 2300 1475, 2330 1445, 2345 1430, 2390 1400, 2405 1385, 2450 1370, 2480 1355, 2510 1340, 2525 1370, 2570 1400, 2585 1430, 2600 1460, 2630 1475, 2645 1505, 2660 1475, 2705 1430, 2735 1400, 2795 1370, 2825 1340, 2855 1385, 2900 1430, 2930 1445, 2945 1475, 2945 1490, 2975 1505, 2990 1475, 3005 1445, 3050 1415, 3080 1400, 3095 1370, 3110 1355, 3155 1340, 3155 1385, 3170 1415, 3200 1445, 3215 1475, 3245 1490, 3260 1505, 3275 1475, 3305 1460, 3350 1415, 3350 1400, 3395 1355, 3410 1355, 3440 1340, 3455 1370, 3470 1400, 3500 1445, 3530 1490, 3560 1505, 3575 1475, 3605 1430, 3665 1400, 3695 1385, 3740 1370, 3770 1340, 3800 1385, 3830 1430, 3875 1475, 3905 1505, 3935 1490, 3965 1445, 3995 1415, 4025 1400, 4055 1370, 4070 1340, 4115 1385, 4130 1415, 4160 1460, 4190 1490, 4205 1505, 4220 1460, 4250 1430, 4265 1400, 4280 1385, 4295 1355, 4310 1355, 4325 1355, 4355 1385, 4385 1415, 4430 1430, 4475 1445, 4505 1460, 4565 1460, 4580 "t2" )2580, 2740 )1430, 1190 1430, 1190 1400, 1205 1385, 1235 1370, 1250 1340, 1295 1130, 1295 1340, 1295 1370, 1295 1400, 1340 1430, 1370 1475, 1415 1505, 1430 1460, 1475 1415, 1520 1370, 1550 1340, 1580 1385, 1595 1400, 1625 1445, 1640 1475, 1670 1490, 1700 1460, 1745 1415, 1760 1400, 1775 1385, 1805 1370, 1820 1340, 1835 1130, 1835 1340, 1835 1385, 1850 1430, 1880 1460, 1910 1475, 1940 1505, 1970 1475, 1985 1445, 2030 1415, 2045 1385, 2090 1370, 2120 1355, 2150 1340, 2180 1370, 2195 1400, 2225 1460, 2255 1490, 2285 1505, 2300 1475, 2330 1445, 2345 1430, 2390 1400, 2405 1385, 2450 1370, 2480 1355, 2510 1340, 2525 1130, 2525 1340, 2525 1370, 2570 1400, 2585 1430, 2600 1460, 2630 1475, 2645 1505, 2660 1475, 2705 1430, 2735 1400, 2795 1370, 2825 1340, 2855 1385, 2900 1430, 2930 1445, 2945 1475, 2945 1490, 2975 1505, 2990 1475, 3005 1445, 3050 1415, 3080 1400, 3095 1370, 3110 1355, 3155 1340, 3155 1130, 3155 1340, 3155 1385, 3170 1415, 3200 1445, 3215 1475, 3245 1490, 3260 1505, 3275 1475, 3305 1460, 3350 1415, 3350 1400, 3395 1355, 3410 1355, 3440 1340, 3455 1370, 3470 1400, 3500 1445, 3530 1490, 3560 1505, 3575 1475, 3605 1430, 3665 1400, 3695 1385, 3740 1370, 3770 1340, 3800 1130, 3800 1340, 3800 1385, 3830 1430, 3875 1475, 3905 1505, 3935 1490, 3965 1445, 3995 1415, 4025 1400, 4055 1370, 4070 1340, 4115 1385, 4130 1415, 4160 1460, 4190 1490, 4205 1505, 4220 1460, 4250 1430, 4265 1400, 4280 1385, 4295 1355, 4310 1340, 4325 1130, 4325 1340, 4325 1355, 4355 1385, 4385 1415, 4430 1430, 4475 1445, 4505 1460, 4565 1460, 4580 "t3" )2580, 2740 "lit" )1430, 1190 1430, 1190 1400, 1205 1385, 1235 1370, 1250 1340, 1295 1190, 1295 1340, 1295 1370, 1295 1400, 1340 1430, 1370 1475, 1415 1505, 1430 1460, 1475 1415, 1520 1370, 1550 1340, 1580 1190, 1580 1340, 1580 1385, 1595 1400, 1625 1445, 1640 1475, 1670 1490, 1700 1460, 1745 1415, 1760 1400, 1775 1385, 1805 1370, 1820 1340, 1835 1190, 1835 1340, 1835 1385, 1850 1430, 1880 1460, 1910 1475, 1940 1505, 1970 1475, 1985 1445, 2030 1415, 2045 1385, 2090 1370, 2120 1355, 2150 1340, 2180 1190, 2180 1340, 2180 1370, 2195 1400, 2225 1460, 2255 1490, 2285 1505, 2300 1475, 2330 1445, 2345 1430, 2390 1400, 2405 1385, 2450 1370, 2480 1355, 2510 1340, 2525 1190, 2525 1340, 2525 1370, 2570 1400, 2585 1430, 2600 1460, 2630 1475, 2645 1505, 2660 1475, 2705 1430, 2735 1400, 2795 1370, 2825 1340, 2855 1190, 2855 1340, 2855 1385, 2900 1430, 2930 1445, 2945 1475, 2945 1490, 2975 1505, 2990 1475, 3005 1445, 3050 1415, 3080 1400, 3095 1370, 3110 1355, 3155 1340, 3155 1190, 3155 1340, 3155 1385, 3170 1415, 3200 1445, 3215 1475, 3245 1490, 3260 1505, 3275 1475, 3305 1460, 3350 1415, 3350 1400, 3395 1355, 3410 1355, 3440 1340, 3455 1190, 3455 1340, 3455 1370, 3470 1400, 3500 1445, 3530 1490, 3560 1505, 3575 1475, 3605 1430, 3665 1400, 3695 1385, 3740 1370, 3770 1340, 3800 1190, 3800 1340, 3800 1385, 3830 1430, 3875 1475, 3905 1505, 3935 1490, 3965 1445, 3995 1415, 4025 1400, 4055 1370, 4070 1340, 4115 1190, 4115 1340, 4115 1385, 4130 1415, 4160 1460, 4190 1490, 4205 1505, 4220 1460, 4250 1430, 4265 1400, 4280 1385, 4295 1355, 4310 1340, 4325 1190, 4325 1340, 4325 1355, 4355 1385, 4385 1415, 4430 1430, 4475 1445, 4505 1460, 4565 1460, 4580 "rep" )3300, 3165 default buttonUp ;buttonUp enterPage default "quest3" "quest4" leavePage leavePage quest3 quest4 Which technique(s) separate on the basis of pI alone?e? Gel filtration Isoelectric focusing 2-Dimensional electrophoresis Ion exchange chromatography SDS polyacrylamide electrophoresiss Which technique(s) separate on the basis of both size and charge? Gel filtration Isoelectric focusing 2-Dimensional electrophoresis Ion exchange chromatography SDS polyacrylamide electrophoresis ethanol By magic antigen binding site quest3 quest4 NextPage 4questscore3 4questscore4 B"3a" B"3b" B"3c" B"3d" B"3e" B"4a" B"4b" B"4c" B"4d" B"4e" default buttonUp buttonUp default questscore4 questscore3 b u y nthe 1430, 1190 1400, 1205 1385, 1235 1370, 1250 1340, 1295 1370, 1295 1400, 1340 1430, 1370 1475, 1415 1505, 1430 1460, 1475 1415, 1520 1370, 1550 1355, 1580 1385, 1595 1400, 1625 1445, 1640 1475, 1670 1490, 1700 1460, 1745 1415, 1760 1400, 1775 1385, 1805 1370, 1820 1355, 1835 1385, 1850 1430, 1880 1460, 1910 1475, 1940 1505, 1970 1475, 1985 1445, 2030 1415, 2045 1385, 2090 1370, 2120 1355, 2150 1340, 2180 1370, 2195 1400, 2225 1460, 2255 1490, 2285 1505, 2300 1475, 2330 1445, 2345 1430, 2390 1400, 2405 1385, 2450 1370, 2480 1355, 2510 1340, 2525 1370, 2570 1400, 2585 1430, 2600 1460, 2630 1475, 2645 1505, 2660 1475, 2705 1430, 2735 1400, 2795 1370, 2825 1340, 2855 1385, 2900 1430, 2930 1445, 2945 1475, 2945 1490, 2975 1505, 2990 1475, 3005 1445, 3050 1415, 3080 1400, 3095 1370, 3110 1355, 3155 1385, 3170 1415, 3200 1445, 3215 1475, 3245 1490, 3260 1505, 3275 1475, 3305 1460, 3350 1415, 3350 1400, 3395 1355, 3410 1355, 3440 1340, 3455 1370, 3470 1400, 3500 1445, 3530 1490, 3560 1505, 3575 1475, 3605 1430, 3665 1400, 3695 1385, 3740 1370, 3770 1340, 3800 1385, 3830 1430, 3875 1475, 3905 1505, 3935 1490, 3965 1445, 3995 1415, 4025 1400, 4055 1370, 4070 1340, 4115 1385, 4130 1415, 4160 1460, 4190 1490, 4205 1505, 4220 1460, 4250 1430, 4265 1400, 4280 1385, 4295 1355, 4310 1355, 4325 1355, 4355 1385, 4385 1415, 4430 1430, 4475 1445, 4505 1460, 4565 1460, 4580 buttonUp buttonUp sysLockscreen "block" "big" 1765,2805 1815, 2280 "small" 1865, 1785 1865,2805 1965, 2280 2065, 1785 1965,2805 2115, 2280 2265, 1785 2065,2805 2265, 2280 2465, 1785 2165,2805 2415, 2280 2665, 1785 2265,2805 2565, 2280 2865, 1785 2365,2805 2715, 2280 3065, 1785 2465,2805 2865, 2280 3265, 1785 2565,2805 3015, 2280 3465, 1785 2665,2805 3165, 2280 3665, 1785 2765,2805 3315, 2280 3865, 1785 2865,2805 3465, 2280 4065, 1785 2965,2805 3615, 2280 4265, 1785 3065,2805 3765, 2280 4465, 1785 3165,2805 3915, 2280 4665, 1785 3265,2805 4065, 2280 4865, 1785 3365,2805 4215, 2280 5065, 1785 3465,2805 4365, 2280 5265, 1785 3565,2805 4515, 2280 5465, 1785 3665,2805 4665, 2280 5665, 1785 3765,2805 4815, 2280 5865, 1785 3865,2805 4965, 2280 6065, 1785 3965,2805 5115, 2280 6265, 1785 4065,2805 5265, 2280 6465, 1785 4165,2805 5415, 2280 6665, 1785 4265,2805 5565, 2280 6730, 1785 4365,2805 5715, 2280 6755, 1785 )870, 2265 default buttonUp buttonUp enterPage block small small small small small small small small small small small small small small small small small small small small small small small small small small small block default B"NextPage" 4questscore20 4questscore19 4questscore18 4questscore17 4questscore16 4questscore15 4questscore14 4questscore13 4questscore12 4questscore11 4questscore10 4questscore9 4questscore8 4questscore7 4questscore6 4questscore5 4questscore4 4questscore3 4count -- If the a question < 5, i.e got something wrong, -- answer that shown. "answer1" "answer2" "answer3" "answer4" "answer5" "answer6" "answer7" "answer8" "answer9" "answer10" "answer11" "answer12" "answer13" "answer14" "answer15" "answer16" "answer17" "answer18" "answer19" "answer20" enterPage leavePage enterPage NextPage answer1 answer1 answer2 answer2 answer3 answer3 answer4 answer4 answer5 answer5 answer6 answer6 answer7 answer7 answer8 answer8 answer9 answer9 answer10 answer10 answer11 answer11 answer12 answer12 answer13 answer13 answer14 answer14 answer15 answer15 answer16 answer16 answer17 answer17 answer18 answer18 answer19 answer19 answer20 answer20 NextPage count questscore1 questscore2 questscore3 questscore4 questscore5 questscore6 questscore7 questscore8 questscore9 questscore10 questscore11 questscore12 questscore13 questscore14 questscore15 questscore16 questscore17 questscore18 questscore19 questscore20 leavePage answer1 answer2 answer3 answer4 answer5 answer6 answer7 answer8 answer9 answer10 answer11 answer12 answer13 answer14 answer15 answer16 answer17 answer18 answer19 answer20 count questscore1 questscore2 questscore3 questscore4 questscore5 questscore6 questscore7 questscore8 questscore9 questscore10 questscore11 questscore12 questscore13 questscore14 questscore15 questscore16 questscore17 questscore18 questscore19 questscore20 "apsrad1" nthe 2850, 1335 2820, 1365 2760, 1410 2700, 1455 2565, 1515 2520, 1530 2475, 1545 2400, 1560 2340, 1560 2220, 1575 2160, 1575 2025, 1590 1875, 1620 1770, 1635 1650, 1665 1560, 1680 1500, 1710 1410, 1695 1350, 1710 1245, 1740 1170, 1755 1065, 1770 960, 1800 870, 1845 780, 1890 675, 1920 570, 1965 510, 1995 420, 2055 330, 2130 240, 2205 165, 2265 120, 2325 120, 2370 135, 2415 150, 2475 210, 2520 240, 2550 270, 2610 345, 2655 420, 2670 435, 2670 480, 2670 540, 2670 630, 2685 675, 2700 750, 2685 810, 2700 buttonUp buttonUp apsrad1 enterPage leavePage enterPage leavePage SDS-PAGE ELECTROPHORESISS FORMATION OF POLYPEPTIDE-SDS COMPLEX (1) Addition of 1% mercaptoethanol to a polypeptide will reduce each disulphide links it contains to two cysteine residues. There is a concomitant unfolding of the structure.... polypeptide chains linked by disulphide bridgee H o _ 1% mercaptoethanolLEEEEEEEEEEEEEEEEEEEEEEEEEEE dimercaptoethanoll polypeptide chains not linked by disulphide bridge sysLockscreen "aps" )2880, 1305 "ac1" )1725, 2790 "ac2" )3555, 2790 "ac3" )5460, 2820 "apsrad1" )2880,1305 "apsrad2" )4095,1305 nthe 2850, 1335 2820, 1365 2760, 1410 2700, 1455 2565, 1515 2520, 1530 2475, 1545 2400, 1560 2340, 1560 2220, 1575 2160, 1575 2025, 1590 1875, 1620 1770, 1635 1650, 1665 1560, 1680 1500, 1710 1410, 1695 1350, 1710 1245, 1740 1170, 1755 1065, 1770 960, 1800 870, 1845 780, 1890 675, 1920 570, 1965 510, 1995 420, 2055 330, 2130 240, 2205 165, 2265 120, 2325 120, 2370 135, 2415 150, 2475 210, 2520 240, 2550 270, 2610 345, 2655 420, 2670 435, 2670 480, 2670 540, 2670 630, 2685 675, 2700 750, 2685 810, 2700 "sul" )570, 2700 "acrad1" )1680, 2490 ZhorizPos -1140 $, 2700 1680 2430 S, 2490 2430, 2520 "polac1" )2580, 2490 2580 3405 , 2490 3405, 2535 "polac2" )3600, 2490 "prop" )90, 3060 "ac4" )9000, 2820 9000 6540 H-100 , 2820 "polac3" )3600, 2490 "ac5" )9000, 2835 9000 7455 H-100 , 2835 "polac4" )3600, 2490 "ac6" )9000, 2835 9000 83550 H-100 , 2835 "polac5" )3600, 2490 default buttonUp buttonUp enterPage apsrad1 apsrad2 apsrad1 apsrad1 apsrad2 acrad1 acrad1 acrad1 acrad1 polac1 polac1 polac1 polac1 polac2 polac2 polac3 polac3 polac4 polac4 polac5 default horizPos:by horizPos:to horizPos 4drop < 12 < + 1) sysLockscreen terPage enterPage leavePage enterPage leavePage &p*.. 4>6h8 :PHYSSIZE Teaching and Learning Technology Programme produced by the Separation Techniques bacteriaah2 Saleem Y Marham & John M Basford Biochemistry Department University of Wales, Cardifffffffffff default buttonUp buttonUp default Start :#~ _ Basic *R*R* You can distribute the unmodified material freely and modify it to your own requirements. However, we ask the following: 1. By all means give yourself credit for your work in your books but please leave this page unaltered in this book. 2. It is important that teaching material of this kind is disseminated as widely as possible, so please ensure that your material is also freely available. 3. Please send a copy of any modified or expanded versions of this program to Dr J.M Basford, Department of Biochemistry, University of Wales, Cardiff, CF1 1ST , Tel 44 222-874119 Fax 44 222-874116. Internet Basford @Cardiff.ac.uk default buttonUp buttonUp default Continue NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 |#T#y# HH 22 n%F%k% NH 22 Z&2&W& HH 22 f(>(c( HH 22 HH 22 NH 22 d,<,a, P-(-M- HH 22 HH 22 HH 22 v0l0s0 NH 22 r3J3o3 HH 22 HH 22 HH 22 NH 22 HH 22 HH 22 b<:<_< HH 22 T>,>Q> NH 22 HH 22 LA$AIA HH 22 HH 22 vDNDsD NH 22 bE:E_E NF&FKF HH 22 nGFGkG HH 22 HH 22 enterPage leavePage enterPage leavePage SDS-PAGE ELECTROPHORESISS FORMATION OF POLYPEPTIDE-SDS COMPLEX (2)) Addition of 1% sodium dodecyl sulphate (SDS) to the unfolded polypeptide will result in a polypeptide-SDS complex being formed. The SDS associates with the polypeptide in such a way as to form an elongated "rod" shape. The binding of SDS imparts a negative charge on the complex, the extent of which is dependent on the mass of the polypeptide - the larger the polypeptides, the higher the charge, giving the same charge/mass ratio.io NaaH2 Sodium dodecyl sulphateEEEEEEEEEEEEEEEEEEEEEEE SDS-polypeptide "rod-shaped" complexCOMPLEXTIDE "ROD" COMPLEX Unfolded polypeptideEEEEEEEEEEEEEEEEEEEEEE sysLockscreen "block" )870, 2265 "elec" )870, 2280 "title" "gel" )1358, 1545 "big" )1665, 2805 )1665, 2280 "small" )1665, 1785 B"animate" "graph" enterPage leavePage enterPage block title small animate graph leavePage nthe 1358, 1545 buttonUp buttonUp title SDS-PAGE ELECTROPHORESISS MOVEMENT OF POLYPEPTIDE-SDS COMPLEXES IN POLYACRYLAMIDE GEL : animate Animate However, when polypeptide-SDS complexes of differing sizes are subjected to electrophoresis in a polyacrylamide gel, the physical barrier imposed by the cross linked polymer prevents them from migrating at the same rate. The smallest complex encounters least resistance and thus, migrates at the fastest rate. Thus SDS-PAGE is a technique which separates SDS-polypeptide complexes on the basis of size alone. ~ N3f block nthe 840, 2310 870, 2265 "big" nthe 1605, 2805 "small" 6945, 2040 6585, 2550 6240, 3000 l'B N V(~ ? ~'B ] 0(` ? sysLockscreen "block" )735, 2085 "gel" B"animate" "glyco" )1680, 1575 "polypep" )1665, 2385 enterPage leavePage enterPage block header animate glyco polypep leavePage AvBvBvBvBvB header J " G SDS-PAGE ELECTROPHORESISS MOVEMENT OF GLYCOPROTEIN-SDS COMPLEXES IN POLYACRYLAMIDE GEL : With glycoprotein-SDS complexes, SDS binds to the polypeptide component only. As a result, there is less charge on the glycoprotein than on a non-glycosylated polypeptide of equivalent mass. Therefore the charge/mass ratio is smaller, and the glycoprotein migrates at a slower rate. An SDS-glycoprotein complex will be more compact than a SDS-protein complex and will tend to more faster for this reason but the dominant effect is that of the reduced charge/mass ratio.o...ffect is that of the reduced charge : mass ratio.s not completely unfolded, and this lessens the effect of the lowered charge : mass ratio somewhat, but not enough to cause equal migration rates. animate Animate nthe 1358, 1545 buttonUp buttonUp ` T.1 block "block" nthe 720, 2085 735, 2085 840, 2310 )735, 2085 polypep "polypep" nthe 1665, 2385 buttonUp buttonUp polypep glyco System Times New Roman Arial Arial Times New Roman Times New Roman Times New Roman Times New Roman Wingdings Times New Roman Wingdings Times New Roman UDUgmJ TECHNIQUES Wingdings System System Times New Roman Times New Roman Arial Times New Roman Times New Roman Times New Roman Times New Roman Times New Roman TimesNewRomanPS TimesNewRomanPS TimesNewRomanPS System Times New Roman Times New Roman Times New Roman System Times New Roman Times New Roman mes New Roman Times New Roman -- Puts the mode hides menus file EnterBook Reader sysRuntime c"Edit" c"Help" c"Text" c"File" sysScreenLock LeaveBook EnterBook LeaveBook EnterBook sizetopage sysScreenLock LeaveBook Basic backPage Previous default buttonUp buttonUp Previous default NextPage default buttonUp buttonUp default ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? FirstPage buttonUp buttonUp 1st Page sysLockscreen "sds" "title" B"animate" "prot1" )3150, 1470 "m1" )-300, 2670 "m2" , 2985 "m3" )4245, -300 "s1" )225, 180 "s2" )1583, 1297 "s3" )3765, -405 "s4" )1013, 2392 "s5" )8040, 120 "s6" )7013, 1597 "s7" )7253, 3157 "s8" )1215, 3990 "s9" )2843, 4777 "s10" )4185, 5220 "s11" )5115, 4620 "s13" )143, 3742 "s12" )7478, 4282 "rod" enterPage leavePage enterPage title animate prot1 leavePage j&"0D; <@<@= prot1 Polypeptide NH3+-- title SDS-PAGE ELECTROPHORESISS FORMATION OF POLYPEPTIDE-SDS COMPLEX (3)) "mcOH1" nthe 3885, 2745 4575, 3060 buttonUp buttonUp mcOH1 mcOH2 "mcOH2" nthe 4620, 1905 buttonUp buttonUp mcOH2 mcOH1 prot2 NH3+-- prot3 NH3+-- N & K b#:#_# prot4 d'<'a' J("(G( NH3+-- L)$)I) P-` O d,<,a, R.*.O. prot5 "prot1" nthe 3150, 1470 "prot2" 3150, 1470 "prot3" 3075, 1485 "prot4" 3075, 1440 "prot5" 1815, 1830 buttonUp buttonUp prot1 prot2 prot3 prot4 prot5 x3P3u3 H4~ & NH3+-- f6>6c6 r8J8o8 N9&9K9 ` $ ~ nthe 4245, 1920 buttonUp buttonUp nthe 3525, 2670 4245, 2985 buttonUp buttonUp "s1" 2250, 3225 "s13" 4703, 3967 "s4" 4148, 2107 "s9" 3083, 3202 "s5" 5250, 1245 "s2" 2453, 2782 "s6" 5558, 2542 "s10" 4140, 3000 "s8" 2670, 3915 "s7" 5558, 3622 "s3" 3525, 2070 "s12" 5273, 3307 "s11" 4995, 1890 buttonUp buttonUp nthe 1583, 1297 1013, 2392 1215, 3990 7013, 1597 7253, 3157 "s12" 7478, 4282 "s1" )225, 180 "s2" )1583, 1297 "s3" )3765, -405 "s4" )1013, 2392 "s5" )8040, 120 "s6" )7013, 1597 "s7" )7253, 3157 "s8" )1215, 3990 "s9" )2843, 4777 "s10" )4185, 5220 "s11" )5115, 4620 "s13" )143, 3742 )7478, 4282 v 3 2J- animate Animate SDS molecules &!V! "F"v" #6#f# #&$V$ %D%t% &4&d& &"'R' (B(r( )0)`) ) *N*~* +>+n+ +.,^, -N-~- .>.n. ../\/ 0L0|0 444d4 4"5R5 "19a" nthe 6570, 1050 "40a" 7365, 1770 "68a" 7470, 2745 "127a" 7065, 3390 "126a" 3750, 4125 "73a" 1965, 4185 "112a" 1365, 3465 "56a" 315, 2220 "20a" 660, 1575 "24a" 1785, 1155 buttonUp buttonUp SDS-PAGE ELECTROPHORESISS GEL FORMATION (3) animate Animate Acrylamide METHYLENE BISACRYLAMIDEEE Methylene bisacrylamide CRYLAMIDEEE )3645, 1680 )4125, 1770 )3270, 3630 )3915, 2115 )4245, 2445 )5700, 2430 )2175, 2565 )2805, 1335 )7650, 3075 )4350, 2130 buttonUp buttonUp l7n7n7 sysLockscreen B"animate" "tewxt" "title" "paper" )90, 1455 "paper2" enterPage leavePage enterPage animate tewxt title paper paper2 leavePage animate tewxt title paper paper2 ,@,p, paper "paper" nthe 90, 1470 buttonUp buttonUp paper 3 W!\ 3 W!\ paper2 "paper" nthe 90, 1470 buttonUp buttonUp paper animate Animate title t2L2q2 ISOELECTRIC FOCUSINGG - SEPARATION BY CHARGE tewxt *6B3'6 Isoelectric focusing is a technique employed to separate mixtures of proteins on the basis of charge alone. The protein is exposed to an electric field in a gel containing pH gradient. Each protein will probably carry some net charge and will move towards one or other electrode. Its charge will alter as it travels through the pH gradient and at some point, at its isoelectric pH (pI), it will carry no net charge. It will therefore stop in the gel at that point and be tightly focused. Size has no effect on the separation.although it will affect the time taken for oteins. The protein mixture is encased in a conducting gel which is subject to an electric field and a consequent pH gradient. Proteins High pH Low pH Intermediate pH Basic Basic backPage Previous default buttonUp buttonUp Previous default NextPage default buttonUp buttonUp default ExitProgram "Really quit?"\ f"Yes" SysSuspendMessages buttonUp buttonUp Really quit? FirstPage buttonUp buttonUp 1st Page sysLockscreen "bloc1" "isogel" "isogel2" )420, 1695 )4215,1740 )4215,1740 )4215,1740 )4455,1740 )4455,1740 4115,1740 4065,1740 3615,1740 4555,1740 4655,1740 4015,1740 3915,1740 3015,1740 4655,1740 4855,1740 3915,1740 3765,1740 2415,1740 4755,1740 5055,1740 3815,1740 3615,1740 1815,1740 4855,1740 5255,1740 3715,1740 3465,1740 1425,1740 4955,1740 5455,1740 U"ff" )1425, 1740 3615,1740 3315,1740 5055,1740 5655,1740 U"fv" )3315, 1740 3515,1740 5155,1740 5855,1740 3415,1740 5255,1740 6055,1740 3315,1740 5355,1740 6255,1740 3215,1740 5455,1740 6455,1740 3115,1740 ,1740 6655,1740 3015,1740 5655,1740 6795,1740 2915,1740 5715,1740 U"fr" )6900, 1740 2815,1740 U"fg" )5835, 1740 2715,1740 2615,1740 2515,1740 2460,1740 U"fb" )2460, 1740 )420, 1785 "t2" )870, 3120 "t3" )870, 3120 "t4" )870, 3120 U"fr" U"fg" U"fb" U"ff" U"fv" "t5" )5340, 2385 "sdsgela" )1140, 1365 "bloc2" )5325, 1380 "sdsgel3" )1140, 1365 "r1" )4545, 1785 "r2" )4545, 1785 "r3" )4545, 1785 "b1" )2040, 1785 "b2" )2040, 1785 "b3" )2040, 1785 "b4" )2040, 1785 "b5" )2040, 1785 "g1" )3900, 1785 "g2" )3900, 1785 "g3" )3900, 1785 "g4" )3900, 1785 "v1" )2565, 1785 "v2" )2565, 1785 "v3" )2565, 1785 "v4" )2565, 1785 "v5" )2565, 1785 "f1" )1545, 1785 "f3" )1545, 1785 "f4" )1545, 1785 "r1" )4545, 1785 "r2" )4545, 1905 "r3" )4545, 2025 "b1" )2040, 1785 "b2" )2040, 1905 "b3" )2040, 2025 "b4" )2040, 2145 "b5" )2040, 2265 "g1" )3900, 1785 "g2" )3900, 1905 "g3" )3900, 2025 "g4" )3900, 2145 "v1" )2565, 1855 "v2" )2565, 1935 "v3" )2565, 2010 "v4" )2565, 2185 "v5" )2565, 2285 "f1" )1545, 1885 "f3" )1545, 2185 "f4" )1545, 2485 "r1" )4545, 1785 "r2" )4545, 2010 "r3" )4545, 2235 "b1" )2040, 1785 "b2" )2040, 1980 "b3" )2040, 2205 "b4" )2040, 2430 "b5" )2040, 2700 "g1" )3900, 1785 "g2" )3900, 1995 "g3" )3900, 2220 "g4" )3900, 2460 "v1" )2565, 1925 "v2" )2565, 2085 "v3" )2565, 2235 "v4" )2565, 2585 "v5" )2565, 2785 "f1" )1545, 1985 "f3" )1545, 2685 "f4" )1545, 3185 "r1" )4545, 1900 "r2" )4545, 2510 "r3" )4545, 3000 "b1" )2040, 1900 "b2" )2040, 2250 "b3" )2040, 2705 "b4" )2040, 3030 "b5" )2040, 3300 "g1" )3900, 1785 "g2" )3900, 1895 "g3" )3900, 2350 "g4" )3900, 2960 "v1" )2565, 2000 "v2" )2565, 2235 "v3" )2565, 2460 "v4" )2565, 2985 "v5" )2565, 3285 "f1" )1545, 2085 "f3" )1545, 2985 "f4" )1545, 3885 "r1" )4545, 2130 "r2" )4545, 3090 "r3" )4545, 3870 "b1" )2040, 2130 "b2" )2040, 2475 "b3" )2040, 3210 "b4" )2040, 3780 "b5" )2040, 4065 "g1" )3900, 1965 "g2" )3900, 2250 "g3" )3900, 2700 "g4" )3900, 3420 "v1" )2565, 2055 "v2" )2565, 2385 "v3" )2565, 2670 "v4" )2565, 3390 "v5" )2565, 3780 "f1" )1545, 2175 "f3" )1545, 3685 "f4" )1545, 4335 )5325, 1380 "t6" )5340, 2385 default "t7" )5340, 2385 buttonUp buttonUp enterPage bloc1 isogel isogel2 bloc1 isogel2 bloc1 sdsgela bloc2 bloc2 sdsgela sdsgel3 bloc2 default sysLockscreen "151" nthe 8130, 3555 "150" 7410, 3585 "149" 6810, 3600 "148" 6210, 3615 "147" 5580, 3570 "146" 4905, 3645 "145" 4350, 3900 "144" 4185, 4365 "143" 3840, 3915 "142" 3600, 4425 "141" 3270, 3945 "140" 3015, 4545 "139" 2895, 4110 "138" 2430, 4455 "137" 2265, 4080 "136" 1845, 4560 "135" 1905, 4200 "134" 1395, 4650 "133" 1380, 4065 "132" 1065, 4530 "131" 345, 4605 "130" 720, 4305 "129" 180, 4050 "128" 780, 3900 "127" 7965, 3090 "126" 7575, 3225 "125" 7110, 3315 "124" 6570, 3255 "123" 5865, 3270 "122" 5925, 2745 "121" 5220, 3090 "120" 4485, 3315 "119" 3975, 3405 "118" 3300, 3405 "117" 2730, 3660 "116" 2085, 3645 "115" 2557, 3038 "114" 1605, 3585 "113" 2055, 3060 "112" 1095, 3345 "111" 570, 3540 "110" 270, 3135 "109" 1545, 3030 "108" 855, 2925 "107" 7440, 2850 "106" 6780, 2805 "105" 5430, 2715 "104" 4755, 2895 "103" 4080, 2940 "102" 3510, 2880 "101" 2505, 2700 "100" 8040, 2415 7425, 2445 6795, 2415 1275, 2580 6405, 2580 5970, 2340 5160, 3405 3045, 2790 2880, 3330 3135, 4920 300, 2580 4365, 2730 2400, 3450 5070, 2640 3825, 2625 5340, 2325 1860, 2715 3345, 2550 4605, 2370 2895, 2460 4005, 2265 2205, 2505 1695, 2430 1200, 2970 1140, 3750 915, 2580 1770, 3330 1695, 3900 3600, 3645 8250, 2805 7125, 2985 5625, 3015 8220, 2085 7785, 2700 5790, 1785 7725, 2055 7095, 2070 6450, 2010 5385, 1980 4995, 2160 4680, 2010 4800, 3255 3690, 3210 675, 3210 600, 2310 3570, 2250 2580, 2310 4200, 1980 3135, 2205 2040, 2235 1125, 2250 7110, 2625 1470, 2130 1275, 1830 870, 1935 495, 1860 165, 2115 8130, 1485 7665, 1695 7245, 1785 6780, 1755 6465, 1515 6135, 1665 5775, 1515 5325, 1665 4995, 1770 4335, 1680 3810, 1905 4665, 1650 3480, 1950 3645, 1605 3135, 1710 2790, 1995 2385, 1935 2010, 1860 1680, 1620 1305, 1455 990, 1515 630, 1425 135, 1530 7695, 1140 7215, 1380 6900, 1140 6450, 1155 5700, 1215 5160, 1335 4680, 1200 4260, 1170 3945, 1410 3600, 1230 3270, 1335 2760, 1350 2430, 1140 2220, 1485 1890, 1215 1395, 1065 930, 1080 480, 1020 135, 1065 enterPage enterPage Buttons Used In The Following Pagess ExitProgram backPage NextPage FirstPage 1st Page Move to the next page Return to previous page Return to the first page of Exit to Windows Animate Animate Animates the sequence of events on that page These are words that are scattered round the text and are shown in italic and bold type and are larger than the surrounding text. They become active when the mouse operated cursor is placed over them. Try pressing this Hotword now! -- Puts the sentence quotation marks a dialog box which can be removed Hclicking "Activating a HOTWORD will present you dthat may contain definitions, references, hints tips, prompts other forms encouragement. Press OK buttonDown buttonDown Activating a HOTWORD will present you with a dialog box that may contain definitions, references, hints and tips, prompts or other forms of encouragement. Press OK to continue Go on to the next page by clicking the button below: Hotwords - sysLockscreen "127a" "126a" "112a" "73a" "68a" "40a" "24a" "20a" "19a" "56a" B"animate" "151" )8130, 3555 "150" )7410, 3585 "149" )6810, 3600 "148" )6210, 3615 "147" )5580, 3570 "146" )4905, 3645 "145" )4350, 3900 "144" )4185, 4365 "143" )3840, 3915 "142" )3600, 4425 "141" )3270, 3945 "140" )3015, 4545 "139" )2895, 4110 "138" )2430, 4455 "137" )2265, 4080 "136" )1845, 4560 "135" )1905, 4200 "134" )1395, 4650 "133" )1380, 4065 "132" )1065, 4530 "131" )345, 4605 "130" )720, 4305 "129" )180, 4050 "128" )780, 3900 "127" )7965, 3090 "126" )7575, 3225 "125" )7110, 3315 "124" )6570, 3255 "123" )5865, 3270 "122" )5925, 2745 "121" )5220, 3090 "120" )4485, 3315 "119" )3975, 3405 "118" )3300, 3405 "117" )2730, 3660 "116" )2085, 3645 "115" )2557, 3038 "114" )1605, 3585 "113" )2055, 3060 "112" )1095, 3345 "111" )570, 3540 "110" )270, 3135 "109" )1545, 3030 "108" )855, 2925 "107" )7440, 2850 "106" )6780, 2805 "105" )5430, 2715 "104" )4755, 2895 "103" )4080, 2940 "102" )3510, 2880 "101" )2505, 2700 "100" )8040, 2415 "99" )7425, 2445 "98" )6795, 2415 "97" )1275, 2580 "96" )6405, 2580 "95" )5970, 2340 "94" )5160, 3405 "93" )3045, 2790 "92" )2880, 3330 "91" )3135, 4920 "90" )300, 2580 "89" )4365, 2730 "88" )2400, 3450 "87" )5070, 2640 "86" )3825, 2625 "85" )5340, 2325 "84" )1860, 2715 "83" )3345, 2550 "82" )4605, 2370 "81" )2895, 2460 "80" )4005, 2265 "79" )2205, 2505 "78" )1695, 2430 "77" )1200, 2970 "76" )1140, 3750 "75" )915, 2580 "74" )1770, 3330 "73" )1695, 3900 "72" )3600, 3645 "71" )8250, 2805 "70" )7125, 2985 "69" )5625, 3015 "68" )8220, 2085 "67" )7785, 2700 "66" )5790, 1785 "65" )7725, 2055 "64" )7095, 2070 "63" )6450, 2010 "62" )5385, 1980 "61" )4995, 2160 "60" )4680, 2010 "59" )4800, 3255 "58" )3690, 3210 "57" )675, 3210 "56" )600, 2310 "55" )3570, 2250 "54" )2580, 2310 "53" )4200, 1980 "52" )3135, 2205 "51" )2040, 2235 "50" )1125, 2250 "49" )7110, 2625 "48" )1470, 2130 "47" )1275, 1830 "46" )870, 1935 "45" )495, 1860 "44" )165, 2115 "43" )8130, 1485 "42" )7665, 1695 "41" )7245, 1785 "40" )6780, 1755 "39" )6465, 1515 "38" )6135, 1665 "37" )5775, 1515 "36" )5325, 1665 "35" )4995, 1770 "33" )4335, 1680 "32" )3810, 1905 "34" )4665, 1650 "31" )3480, 1950 "30" )3645, 1605 "29" )3135, 1710 "28" )2790, 1995 "27" )2640, 1590 "26" )2385, 1935 "25" )2010, 1860 "24" )1680, 1620 "23" )1305, 1455 "22" )990, 1515 "21" )630, 1425 "20" )135, 1530 "19" )7695, 1140 "18" )7215, 1380 "17" )6900, 1140 "16" )6450, 1155 "15" )5700, 1215 "14" )5160, 1335 "13" )4680, 1200 "12" )4260, 1170 "11" )3945, 1410 "10" )3600, 1230 )3270, 1335 )2760, 1350 )2430, 1140 )2220, 1485 )1890, 1215 )1395, 1065 )930, 1080 )480, 1020 )135, 1065 sysLocksreen enterPage leavePage enterPage animate sysLocksreen leavePage sysLocksreen Basic Basic enterPage leavePage enterPage leavePage SDS-PAGE ELECTROPHORESISS GEL FORMATION (1) Polyacrylamide gels are formed by the polymerisation of acrylamide and methylene bisacrylamide (MBA) molecules. Acrylamide polymerises to form long chains but the incorporation of occasional MBAs creates crosslinks between chains. The result is a 3-dimensional mesh. The pores of this mesh are similar in size to macromolecules and the mesh thus acts as a molecular sieve. When molecules are forced to move through the gel by a force such as an electric field, the gel will present more resistance to the movement of large molecules than to that of small ones.s dependent on several factors AcrylamideeE Methylene bisacrylamideeEEEEEEEEEEEEEEEEEE NH 22 NH222 NH222