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
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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.
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answer20
"answer20"
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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"
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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"
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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"
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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"
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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
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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"
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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.
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BIBLIOGRAPHY
Pharmacia- ION EXCHANGE CHROMATOGRAPHY
Pages 4-7 7 - 109555555555555
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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.
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The proteins are thus separated on the basis of both size and charge. dimensions.
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polyacrylamide gel
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isoelectric focusing gel roddraperfocussing paper
The gel is then infused with SDS and mercaptoethanol,.e.
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title
2-DIMENSIONAL ELECTROPHORESISSS
-A POWERFUL SEPARATION TECHNIQUEE
IN POLYACRYLAMIDE GEL :
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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.
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SDS-polyacrylamide gel electrophoresis is then performed in the second dimension .....
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REPEAT OR MOVE TO NEXT PAGEEE..........basis of both size and charge. dimensions.
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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.....
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DNA ELECTROPHORESISSSSSSSSSSSSSS
USING POLYACRYLAMIDE OR AGAROSE ::::::::::::::::::::::::::::::
o &54
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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 :
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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
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rband
rband
rband
rbeek
rbeek
mixband
gband
gband
gband
gbeek
gbeek
pband
pband
pband
pbeek
pbeek
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"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"
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column
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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
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rbeek
nthe
315, 1650
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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
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column
"rtip"
nthe
3390, 3330
buttonUp
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pband
"pband"
nthe
3390, 2640
3375, 1845
3390, 3210
rband
gband
mixband
of selection to 750
title
)570, 285
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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"
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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
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4questscore5
4questscore6
B"5a"
B"5b"
B"5c"
B"5d"
B"5e"
B"6a"
B"6b"
B"6c"
B"6d"
B"6e"
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questscore6
questscore5
"quest7"
"quest8"
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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
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4questscore8
B"7a"
B"7b"
B"7c"
B"7d"
B"7e"
B"8a"
B"8b"
B"8c"
B"8d"
B"8e"
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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
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"quest9"
"quest10"
leavePage
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quest9
quest10
Polymerisation of which two molecules creates polyacrylamide?
Persulphate
Acrylamide
Ethylene bisacrylamide
Bismethylene acrylamide
Methylene bisacrylamidee
quest9
quest10
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4questscore10
B"9a"
B"9b"
B"9c"
B"9d"
B"9e"
B"10a"
B"10b"
B"10c"
B"10d"
B"10e"
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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
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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
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4questscore11
4questscore12
B"11a"
B"11b"
B"11c"
B"11d"
B"11e"
B"12a"
B"12b"
B"12c"
B"12d"
B"12e"
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questscore12
questscore11
sysLockscreen
B"animate"
"aps"
)2880, 1305
"ac1"
)1725, 2790
"ac2"
)3555, 2790
"ac3"
)5460, 2820
"title"
sysLocscreen
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animate
title
sysLocscreen
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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
b<X<_<
acrad1
NH222
z@R@w@
HH 22
NA&AKA
HH 22
HH 22
PCFCMC
polac3
NH 22
HH 22
jIBIgI
HH 22
HH 22
NH 22
nNFNkN
HH 22
HH 22
HH 22
bR:R_R
NH 22
TS,SQS
HH 22
HH 22
HH 22
nthe
2580, 2505
buttonUp
buttonUp
LY$YIY
NH 22
HH 22
t\L\q\
HH 22
HH 22
X^N^U^
polac1
"acrad2"
nthe
3510, 2520
3510, 2505
buttonUp
buttonUp
acrad2
Tb,bQb
NH 22
HH 22
|eTeye
HH 22
HH 22
nthe
2580, 2505
buttonUp
buttonUp
NH 22
HH 22
fl>lcl
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"
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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
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questscore14
questscore13
quest13
quest14
"quest15"
"quest16"
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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"
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questscore16
questscore15
quest15
quest16
"quest17"
"quest18"
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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"
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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
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enterPage
matrix2
animate
title
FirstPage
ExitProgram
backPage
NextPage
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"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
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the answer boxes
"quest1"
"quest2"
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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
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question 1. It
zero
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questscores 2-20
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fquestions on
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4questscore2
B"1a"
B"1b"
B"1c"
B"1d"
B"1e"
B"2a"
B"2b"
B"2c"
B"2d"
B"2e"
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4drop
< 42
< + 1)
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terPage
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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.
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THE END
FirstPage
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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.
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"gel"
"big"
"lit"
"inv"
"permtext"
B"test"
"title"
"rep"
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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
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nthe
2625, 3300
2610, 3480
"117"
2505, 3630
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1950, 2520
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1920, 1890
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4245, 1800
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5430, 1905
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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
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1475, 2645
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1385, 4130
1415, 4160
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1370, 1550
1340, 1580
1385, 1595
1400, 1625
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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
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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
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1370, 4070
1340, 4115
1385, 4130
1415, 4160
1460, 4190
1490, 4205
1505, 4220
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1385, 4295
1355, 4310
1340, 4325
1130, 4325
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1460, 4580
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1340, 1580
1190, 1580
1340, 1580
1385, 1595
1400, 1625
1445, 1640
1475, 1670
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1460, 1745
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1370, 1820
1340, 1835
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1385, 1850
1430, 1880
1460, 1910
1475, 1940
1505, 1970
1475, 1985
1445, 2030
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1370, 2120
1355, 2150
1340, 2180
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1340, 2180
1370, 2195
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1385, 2900
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1385, 3170
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1505, 3275
1475, 3305
1460, 3350
1415, 3350
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1355, 3410
1355, 3440
1340, 3455
1190, 3455
1340, 3455
1370, 3470
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1445, 3530
1490, 3560
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1430, 3665
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1385, 3740
1370, 3770
1340, 3800
1190, 3800
1340, 3800
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1475, 3905
1505, 3935
1490, 3965
1445, 3995
1415, 4025
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1370, 4070
1340, 4115
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1385, 4130
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"quest3"
"quest4"
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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
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B"3a"
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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
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1370, 4070
1340, 4115
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2165,2805
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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"
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"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
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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
$, 2700
1680
2430
S, 2490
2430, 2520
"polac1"
)2580, 2490
2580
3405
, 2490
3405, 2535
"polac2"
)3600, 2490
"prop"
)90, 3060
"ac4"
)9000, 2820
9000
6540
H-100
, 2820
"polac3"
)3600, 2490
"ac5"
)9000, 2835
9000
7455
H-100
, 2835
"polac4"
)3600, 2490
"ac6"
)9000, 2835
9000
83550
H-100
, 2835
"polac5"
)3600, 2490
default
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apsrad1
apsrad2
apsrad1
apsrad1
apsrad2
acrad1
acrad1
acrad1
acrad1
polac1
polac1
polac1
polac1
polac2
polac2
polac3
polac3
polac4
polac4
polac5
default
horizPos:by
horizPos:to
horizPos
4drop
< 12
< + 1)
sysLockscreen
terPage
enterPage
leavePage
enterPage
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&p*..
4>6h8
:PHYSSIZE
Teaching and Learning Technology Programme
produced by the
Separation Techniques
bacteriaah2
Saleem Y Marham & John M Basford
Biochemistry Department
University of Wales, Cardifffffffffff
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Start
:#~ _
Basic
*R*R*
You can distribute the unmodified material freely and modify it to your own requirements. However, we ask the following:
1. By all means give yourself credit for your work in your books but please leave this page unaltered in this book.
2. It is important that teaching material of this kind is disseminated as widely as possible, so please ensure that your material is also freely available.
3. Please send a copy of any modified or expanded versions of this program to Dr J.M Basford, Department of Biochemistry, University of Wales, Cardiff, CF1 1ST , Tel 44 222-874119 Fax 44 222-874116.
Internet Basford @Cardiff.ac.uk
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Continue
NH 22
HH 22
HH 22
HH 22
NH 22
HH 22
HH 22
HH 22
NH 22
HH 22
HH 22
HH 22
NH 22
HH 22
HH 22
|#T#y#
HH 22
n%F%k%
NH 22
Z&2&W&
HH 22
f(>(c(
HH 22
HH 22
NH 22
d,<,a,
P-(-M-
HH 22
HH 22
HH 22
v0l0s0
NH 22
r3J3o3
HH 22
HH 22
HH 22
NH 22
HH 22
HH 22
b<:<_<
HH 22
T>,>Q>
NH 22
HH 22
LA$AIA
HH 22
HH 22
vDNDsD
NH 22
bE:E_E
NF&FKF
HH 22
nGFGkG
HH 22
HH 22
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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
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
8=` N
^=` ?
~ zp
small
"big"
nthe
1665, 3105
1680, 2580
"small"
1710, 2085
buttonUp
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small
graph
Log Mr
Mobility
HZIFK2M
RhXTZ@\,^
nZtFv2x
matrix2
nthe
-105, -195
-135, -15
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MATRIX BEAD CONTAINING
CATIONIC GROUPS
MATRIX BEAD CONTAINING
CATIONIC GROUPS
j0B0g0
Phosphate counter ion
Chloride elution ionn
"p2"
3830, 1295
"p2"
3980, 1460
"p2"
4130, 1715
"p2"
4205, 2030
"p2"
4280, 2360
"p2"
4295, 2930
"p2"
4295, 3245
"p2"
4325, 3620
"p2"
4340, 3950
"p2"
4355, 4655
"p2"
4385, 5375
nthe
4190, 1040
4190, 1250
4175, 1445
4160, 1595
4130, 1805
4025, 1820
3920, 1850
3815, 1865
3665, 1865
4250, 965
4400, 1070
4475, 1160
4565, 1205
4625, 1235
4715, 1355
4070, 1070
3950, 1130
3800, 1145
3680, 1145
4175, 890
3850, 4930
4120, 5275
4315, 5875
3655, 4825
nthe
4430, 1715
4280, 1955
4235, 2195
4220, 2600
4220, 3185
4190, 3905
4145, 4760
buttonUp
buttonUp
nthe
3785, 1985
4010, 2570
4025, 3035
4145, 4085
4130, 5315
3710, 2030
3860, 2240
3980, 2480
3965, 2780
3995, 3200
3995, 3740
3995, 4040
4025, 4880
4085, 5480
4165, 4600
4075, 4630
3925, 4690
3835, 4735
3730, 4810
3655, 4825
4270, 3250
4315, 3280
4420, 3340
4510, 3370
4600, 3385
4630, 3430
4660, 3430
3985, 1660
3910, 1735
3760, 1840
3700, 1900
3655, 1885
3655, 1885
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FirstPage
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Initially, proteins of different anionic charge bind to the cations on the matrix.
nthe
4410, 1185
4035, 1680
4680, 2130
3975, 2325
4485, 2550
4005, 3105
4365, 3540
3840, 4065
4050, 4455
4815, 4575
3990, 4740
4410, 5010
3975, 5190
4845, 5505
3870, 5640
buttonUp
buttonUp
nthe
4575, 2115
3915, 1455
4560, 1605
4155, 2025
4050, 1740
3765, 3645
4020, 3540
3765, 4530
3945, 5145
4005, 5385
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nthe
4815, 1140
4215, 1485
3675, 1380
4455, 1890
4365, 2310
4845, 2520
4290, 2865
3675, 3630
4215, 3270
4485, 3780
4050, 3870
4560, 4095
3705, 4470
4365, 4410
4260, 4785
4710, 5025
4260, 5265
4005, 5445
4560, 5295
buttonUp
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"3rep"
nthe
4835, 1400
buttonUp
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nthe
3990, 1215
4620, 1605
4215, 2055
4050, 2715
4560, 3105
3975, 3525
4245, 4140
4515, 4650
4335, 5535
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animate
Animate
When an elution buffer of a certain concentration is used, the protein with the lowest charge is eluted.
Increasing the concentration of the buffer will cause elution of the remaining proteins in the order of their charge.
REPEAT OR MOVE
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PRESS THE
ANIMATE BUTTON
title
"title"
nthe
570, 285
buttonUp
buttonUp
title
ION EXCHANGE CHROMATOGRAPHYYSSS
-AT THE MOLECULAR LEVELEEEEEEEEEE
IN POLYACRYLAMIDE GEL :
sysLockscreen
"block"
)885, 2235
"elec"
)885, 1605
B"animate"
"title"
"small"
)1665, 1785
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block
animate
title
small
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title
SDS-PAGE ELECTROPHORESISS
MOVEMENT OF POLYPEPTIDE-SDS COMPLEXES
IN FREE SOLUTION:
animate
sysLockscreen
"block"
ZhorizPos
1665
"small"
), 1785
)885, 2235
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block
small
block
default
horizPos:by
horizPos:to
horizPos
Animate
When polypeptide-SDS complexes of differing sizes are subjected to electrophoresis in free solution, they all migrate at the same rate. This is because rate of migration depends on the charge/mass (q/m) ratio, and this is constant for polypeptide-SDS complexes of all sizes. SDS binds to polypeptides proportionally to mass, ie, the larger the polypeptide, the more SDS binds. This is how SDS binding maintains q/m ratio.
block
"block"
)885, 2235
buttonUp
buttonUp
block
small
>'B N
V(~ ?
~'B ]
0(` ?
sysLockscreen
"block"
)735, 2085
"gel"
B"animate"
"glyco"
)1680, 1575
"polypep"
)1665, 2385
enterPage
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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
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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
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menus
file
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Basic
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FirstPage
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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
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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
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mcOH1
mcOH2
"mcOH2"
nthe
4620, 1905
buttonUp
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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
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prot1
prot2
prot3
prot4
prot5
x3P3u3
H4~ &
NH3+--
f6>6c6
r8J8o8
N9&9K9
` $ ~
nthe
4245, 1920
buttonUp
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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
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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
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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
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paper
3 W!\
3 W!\
paper2
"paper"
nthe
90, 1470
buttonUp
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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
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FirstPage
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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
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Basic
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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
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