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 

ACIDIC-NATIVE GEL Protocol
For Basic Proteins (pI>7.0)
USE ONLY FRESH GELS

Stock Solutions
1) 1.5M Acetate-KOH pH 4.3 (48ml 1MKOH + 17.2ml AcH + H2O up to 200ml) - Keep RT.
2) 30% Acrylamide 0.8% Methylene bis Acrylamide. Keep 4°C.
3) 0.25M Acetate-KOH pH 6.8 (48ml 1MKOH + 2.9ml AcH + H2O up to 200ml) - Keep RT.
4) 10% Ammonium Persulfate (APS). Keep 4°C less than 1 month.
 
Running Buffer x1
 
ß-Alanine
0.35M
18.7gr
AcH
0.14M 
4.8ml 
H2O up to

600ml
Adjust pH to
 
4.3
Keep at RT


Sample Buffer x5
 
Glycerol 50%
1.45ml
0.25M Acetate-KOH pH 6.8 
0.5ml
Methyl Green
traces

Keep in aliquots of 1ml at -20C
 
 

Separating Gel

 
% Acrylamide
10%
15%
7%


Gradient 7.5%
Gradient 20%
1.5M Acetate-KOH pH 4.3
6.7 ml
6.7 ml
6.7 ml


5.25ml
5.25ml
50% Glycerol
6.0ml 6.0ml 6.0ml

0ml
4.20ml
30% Acrylamide 0.8% Methylene bis Acrylamide
8.8 ml
13.3 ml
6.4 ml


5.25ml
14.0 ml
H2O
4.2 ml
0 ml
6.6 ml


2.9 ml
0 ml
10% APS
320 ul
320 ul
320 ul


160 ul
80 ul
TEMED
40 ul
40 ul
40 ul


50 ul
50 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 H2O before pouring the stacking gel.
 

Stacking Gel
 
Number of Minigels
2
5
8
0.25M Acetate-KOH pH 6.8 
2.5 ml
4.0 ml
5.0 ml
30% Acrylamide 0.8% Methylene bis Acrylamide
1.0 ml
1.5 ml
2.0 ml
H2O
6.4 ml
9.6 ml
12.8 ml
10% APS
100 ul
150 ul
200 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. Keep gel at 4C no more than 2 days; use only fresh gels. REVERSE POLARITY OF THE LEADS TO THE GEL(At an acidic pH the proteins will be positively charged, and therefore run toward the cathode, so if we do not reverse the polarity the sample will be lost!). Running conditions: 40mA / 250V max.

Sample Preparation

Prior to adding the sample buffer, keep samples at 0°C. Add the sample buffer (RT) to the sample (still on ice), and load immediately.
For a gel thickness of 0.75mm and 15 wells applied 0.5 to 5ug for Coomasie Blue stain and 10 to 100-fold less protein for silver stain.
If you do not know the electrophoretic pattern of your protein, load same sample in parallel wells at different times during the run.
Use only purified or paritally purified material.
 

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 few seconds in microwave on the high position, and then shake for another 15min at RT. Wash with water 2-3 times and destain with 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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