Lecture notes on the topic Protein Electrophoresis, taught by Professor Steve Matthews at Imperial College London. Course: Bsc Biochemistry. Module: Structural Biology.
Protein Electrophoresis
↳
migration of proteins in an applied electric field
-
free flow electrophoresis 1 diffusion in solution ) is feasible , but convection broadens band and
limits resolution
-
most kinds of electrophoresis is carried out in a polymeric medium I i. e. gets )
↓
diffusional broadening still present but reduced as molecules diffuse slower in gets
-
migration rate ( M) is proportional to : electric field strength ,
ionic strength ,
net charge , temperature ,
molecular size & shape ,
viscosity of sample
Types of protein electrophoresis : 1 . Under denaturing conditions I dependent on size)
2. Under native conditions I dependent on size and charge )
3. Isoelectric focussing IIEF ) ( dependent on PI )
4. 2 -
dimensional ( dependent on PI in 1 dimension and
size in the 2nd dimension 1
Polymer phase I get )
↳ and crosslinked with
usually acrylamide , polymerized into polyacrylamide ,
methylene bis -
acrylamide
-
pore size is determined by [ acrylamide] and the ratio of
acrylamide :
methylene bis -
acrylamide
if large pore get used 15% acrylamide ) there is
-
,
polyacrylamide no molecular weight sieving of proteins < 100 kDa
crosslink
as [ acrylamide ] ↑ 110-15%7 sewing range ↑
-
,
-
detection and visualisation : coomassie blue dye
1- 1mg of protein per band )
or silver staining 11 ng)
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- in presence of p mercapto ethanol
-
1 reducing agent 7 and
sodium dodecyl sulphate 1 anionic detergent )
V
V. hydrophobic , form micelles ,
binds to hydrophobic core of protein
L
multi subunit proteins dissociate , polypeptide chains unfold and bind SDS on a
constant weight basis 11.4g SDS /
g of peptide )
protein intrinsic charge swamped by the charge SDS
'
is
'
- -
ve on
: all proteins have the same charge density
i.
only migrate according to size I not charge)
-
resolution improved by using discontinuous gets
>
5% acrylamide ,
Tris / HCl buffer at PH 6.7
electric field runs vertically ↳ no molecular weight
sieving
direction
10 -15% acrylamide ,
glycine is also ✓ Tris / HCl buffer at pH 8.9
in the buffer ↳ molecular weight sieving
* if manual process of loading wells is done poorly ,
resolution will not be affected
( negative impact for continuous gets )
proteins ifncentrate in stacking get first
glycine has PI Of -6
it "
°
↳
stacking get has pH 6.7 I close -10 PI ) H -
C -
C
'
I
18
-
small net ve) o
: -
ve
charge -
+
NH
}
1 above PI )
↳
running get has pH 8.9
it "
0
:
large -
ve charge H C-
-
C all ionisable groups
'
1 -
0 are deprotonated
✓ NHZ
high mobility
applied voltage proportion of charged molecules
equation determining migration rate rate V IS Mn )
✗ effective mobility
: =
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