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