Agarose plug: 
1% agarose dissolved in 1x Resolving gel buffer. 
（I make 50 ml, keep melting it as I need it, and re-adding water to maintain agarose conc.） 
Resolving gel: 24 ml of a 9% gel 
5.4 ml 40% acrylamide/bisacrylamide （29:1 mix） 
3 ml 8x Resolving gel buffer 
15.6 ml water 
12 µl TEMED 
60 µl 20% ammonium persulfate 
Stacking gel: 8 ml 
1 ml 40% acrylamide/bisacrylamide （29:1 mix） 
2 ml 4x Stacking gel buffer 
5 ml water 
8 µl TEMED 
21.6 µl 20% ammonium persulfate 

C. Preparation of gel 

Assemble the glass plates and spacers （1.5 mm thick）. 
Pour an agarose plug （1-2 mm）. 
Pour the running gel to about 1 cm below the wells of the comb （~20 ml）. 
Seal with 1 ml water-saturated 1-butanol. 
（Can stop here and leave gel as is overnight if you want.） 
When gel has set, pour off the butanol and rinse with deionized water. 
Pour the stacking gel （~5 ml） and insert the comb immediay. 
When the stacking gel has set, place in gel rig and immerse in buffer. 
Prior to running the gel, flush the wells out thoroughly with running buffer. 

D. Running the gel 

After flash spinning the samples, load into the wells. 
Be sure to use markers. 
We use 15 µl Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly. 
Run with constant current （35 - 37 mA with voltage set at > 300 V）. 
Usual running time is about 2.5 hr. 

E. Using precast gels （Ready Gels from Bio-Rad）: 

Assemble gel in gel rig. 
Prepare protein samples （10 µg will suffice）. 
Use 5 µl of Kaleidoscope standard. 
Run at 200 V （constant voltage） for 30 min. 

F. Preparation of membrane 

Cut a piece of PVDF membrane （Millipore Immobion-P #*H 000 10）. 
Wet for about 30 min in methanol on a rocker at room temp. 
Remove methanol and add 1x Blotting buffer until ready to use. 

G. Membrane transfer