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