The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920 

PAGE-SDS Laemmli Protocol

Stock Solutions
1) 1.5M TrisHCl pH 8.3 + 0.4% SDS (adjust pH before you add the SDS). Keep RT.
2) 30% Acrylamide 0.8% Methylene bis Acrylamide or 40% Acrylamide / Methylene bis Acrylamide (ratio: 37.5:1). Keep 4°C
3) 0.5M TrisHCl pH 6.8+ 0.4% SDS (adjust pH before you add the SDS). Keep RT.
4) 10% Ammonium Persulfate (APS). Keep 4°C less than 1 month.
 
Running Buffer x5
 
Trizma
0.125M
15.1gr
Glycine
0.96M 
72.0gr 
SDS
0.5%
5.0gr
H2O up to
 
1 liter
Keep at RT


Dissolving (Sample) Buffer x5
 
Glycerol
5ml
SDS
1gr
ß Mercapto Ethanol
(or 0.25M DTT)
2.56ml
0.5M TrisHCl pH 6.8+ 0.4% SDS 
2.13ml
Bromo Phenol Blue
traces
Keep in aliquots of 1ml at -20C

 

Separating Gel


 
% Acrylamide
10%
10%
12%
12%
15%
Number of Minigels
5
8
5
8
5
1.5M TrisHCl pH 8.3 + 0.4% SDS
7.0 ml
10.5 ml
7.0 ml
10.5 ml
7.0 ml
30% Acrylamide 0.8% Methylene bis Acrylamide
9.3 ml
13.9 ml
11.3 ml
16.9 ml
13.9 ml
H2O
12.3 ml
18.4 ml
9.3 ml
13.9 ml
6.3 ml
10% APS
100 ul
150 ul
100 ul
150 ul
100 ul
TEMED
23 ul
35 ul
23 ul
35 ul
23 ul

 
% Acrylamide
10%
10%
12%
12%
15%
Number of Minigels
5
8
5
8
5
1.5M TrisHCl pH 8.3 + 0.4% SDS
7.0 ml
10.5 ml
7.0 ml
10.5 ml
7.0 ml
40% Acrylamide / Methylene bis Acrylamide (ratio: 37.5:1)
7.2 ml
10.8 ml
8.6 ml
12.9 ml
10.8 ml
H2O
14.4 ml
21.5 ml
12 ml
17.9 ml
9.4 ml
10% APS
100 ul
150 ul
100 ul
150 ul
100 ul
TEMED
23 ul
35 ul
23 ul
35 ul
23 ul

 

Add TEMED and APS at the end. Gently swirl the flask to mix, being careful not to generate bubbles. Pipette the solution to a level of 4cm of the top. Add 0.3ml of n-buthanol. A very sharp liquid interface will be visible within 10-20min. Let polymerize the gel for another hour at least. Rinse the surface of the gel with watter before pouring the stacking gel.
 
 

Stacking Gel
 
Number of Minigels
2
5
8

Number of Minigels
2
5
8
0.5M TrisHCl pH 6.8+ 0.4% SDS 
2.5 ml 4.0 ml 5.2 ml  
0.5M TrisHCl pH 6.8+ 0.4% SDS 
2.5 ml
4.0 ml
5.2 ml
30% Acrylamide 0.8% Methylene bis Acrylamide
1.0ml
1.5ml
2.0ml
 
40% Acrylamide / Methylene bis Acrylamide (ratio: 37.5:1)
0.75 ml
1.1 ml
1.4 ml
H2O
6.4ml
9.6ml
12.8ml
 
H2O
6.6 ml
10 ml
13.4ml
10% APS
100 ul
150 ul
200 ul
 
10% APS
100 ul
150 ul
200 ul
TEMED
10 ul
15 ul
20 ul
 
TEMED
10 ul
15 ul
20 ul

Fill each sandwich with stacking gel solution and insert a comb into each place taking care not to trap any bubbles bellow the teeth. The gel should fully polymerized after 1hour. Cover  gel with a nylon wrap.  Keep gels no more than 2 weeks at 4°C.
 

Sample Preparation

Prior to adding the sample buffer, keep samples at 0°C. Add the SDS sample buffer (RT) to the sample (still on ice), and boil at 100°C immediately 3 to 5 min. DO NOT leave the sample in SDS sample buffer without heating; endogenous proteases are very active in SDS sample buffer and can cause severe degradation. Once heated, sample could sit at RT for a short time until loading, or at -20°C for a long time.
For a gel thickness of 0.75mm and 15 wells applied 10-25ug protein of a complex mixture, when staining with Coomasie Blue and 0.5 to 5ug for samples for one or few proteins. If silver stain is used 10 to 100-fold less protein can be used.
Samples can be concentrated or interferences (salts, etc.) eliminated with TCA, acetone, TCA-DOC, ethanol, etc. (see attached Protocol). Potassium ions in particular must be removed since they precipitate the SDS.
Some proteins such as nuclear non-histone proteins and membrane proteins, require the presence of 8M urea in the SDS sample buffer to get complete solubilization.
Some membrane bound proteins undergo aggregation at temperatures above 40-50°C. In this case incubate 30min at 40°C with sample buffer.
A shift in the migration distances of proteins 
with internal disulfide bridges could be observed by incubating samples in SDS in the presence or absence of reducing agents (mercaptoethanol, DTT, DTE, etc)


 
 

Staining Solution
 
Methanol CP 
500ml
50%
Acetic Acid CP
100ml
10%
H2O
400ml
Coomasie Brilliant Blue R
2.5gr
0.25%

Keep flask on dark at RT

Destaining Solution
 
Methanol CP 
150ml
15%
Acetic Acid CP 
100ml
10%
H2O
750ml

Keep flask on dark at RT

 
Stain overnight at RT or put gel with staining solution 8 sec in microwave on the high position, and then shake for another 15min at RT. Wash with water 2-3 times and distain by several changes of destaining solution in the presence of a sponge.
HOME
EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS



Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@mail.ls.huji.ac.il  Tel: 972-2-6586920  

Copyright ©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.