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PAGE-SDS Laemmli Protocol
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 

TRICINE GELS Protocol
(Shagger H. et al., 1987, Anal Biochem.166: 368-379 and SDS Electrophoresis Techniques - Memebrane Protein Purification and Crystallization 2/e: A Practical Guide, 2003, Elsevier Science).
For Low MW proteins (<25000) and peptides



Stock Solutions
1) Gel Buffer: 3 M TrisHCl pH8.45 + 0.3% SDS. Keep RT. (Prepare: 18.15gr Tris + 150mg SDS  + H2O up to 50ml. Adjust pH before you add the SDS)
2) 40% Acrylamide solution 29:1 (Acrylamide 29 / Methylene bis Acrylamide 1) or 40% Acrylamide solution 19:1 (Acrylamide 19 / Methylene bis Acrylamide 1) for short peptides. Keep 4°C.
3) Glycerol 70%
4) Cathode buffer 10X (upper buffer): 1M Tris + 1M Tricine + 1% SDS pH 8.25. Keep RT. (Prepare: 24.2gr Tris + 35.84gr Tricine       + 2gr SDS +H2O up to 200ml. Adjust pH 8.25 before you add SDS).
5) Anode buffer 10X (lower buffer): 2.1M Tris pH8.9 Keep RT. (Prepare: 48.4gr Tris. Adjust pH 8.9 and H2O up to 200ml).
6) 10% Ammonium Persulfate (APS). Keep less than one month at 4°C.
7) TEMED
 
 
 
Sample Buffer x5
 
Glycerol
5ml
SDS
1gr
ß Mercapto Ethanol
(or 0.25M DTT)
2.56ml
0.5M TrisHCl pH 6.8 
2.13ml
Bromo Phenol Blue
traces

Keep in aliquots of 1ml at -20C
 
 



Separating and Stacking Gel
SOLUTIONS
Separating gel
Short gels: 4
Large gels: 2
Separating gel
Short gels: 2
Stacking gel
Short gels: 4
Stacking gel
Short gels: 2
Acrylamide Solution
10 ml
 5.00 ml
2.12 ml 
1.05 ml 
Gel Buffer
10 ml
5.00 ml
5 ml
2.5 ml
Glycerol 70%
4 ml
2.00 ml
0 ml
0 ml
H2O
6.2 ml
3.1 ml
13.44 ml
6.70 ml
10% APS
133 µl
66 µl
160 µl
80 µl
TEMED
13.2 µl
6.6 ul
16  µl
8 µl

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 H2O before pouring the stacking gel.

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 wrap nylon.  Keep gels no more than 2 weeks at 4°C. Running conditions: 200 Volts constant.


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)
Running conditions:  Start running at 60mA 200V 10minutes (to get samples inside stacking gel), and then continue at 35mA 200V
 

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.
 
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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  

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