The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920 


For Low MW proteins (<25000) and peptides

Shagger H. et al., (1987), Anal Biochem.166: 368-379 and SDS Electrophoresis Techniques - Memebrane Protein Purification and Crystallization
        A Practical Guide, 2003, Elsevier Science.

Shagger H  (2006)  NATURE PROTOCOLS Vol  1  Number 1: 16-23   Tricine-SDS- PAGE  (pdf) 

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.
Sample Buffer x5
ß Mercapto Ethanol
(or 0.25M DTT)
0.5M TrisHCl pH 6.8 
Bromo Phenol Blue

Keep in aliquots of 1ml at -20C

Separating and Stacking Gel
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
6.2 ml
3.1 ml
13.44 ml
6.70 ml
10% APS
133 µl
66 µl
160 µl
80 µl
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 
Acetic Acid CP
Coomasie Brilliant Blue R

Keep flask on dark at RT

Destaining Solution
Methanol CP 
Acetic Acid CP 

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.


Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,   The Hebrew University of Jerusalem  Tel: 972-2-6586920  

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