Hello Kunal:
I have been running protein gels for years .. but I never had such a
problem!!! Couple of things you should be careful:
Impurities in protein sample (I normally ppt proteins with 10% TCA, wash with
80% Acetone, air dry and dissolve in SDS-PAGE loading buffer, pH 6.9.. if
there are indeed traces of TCA.. the loading buffer turns yellow.. in that
case add 1/10 vol. of 2M Tris base to neutralize the sample).. heat denature
before loading.
As far as cleaning plates and pouring gel... Well I do not wash plates with
distilled water after ethanol.. it is not necessary.. besides if you
observe.. washing plates with EtOH will keep the plates dust free and also
the gel will not stick to the glass plate!!
Try this recipe:
Separating gel (I have run many gels upto 15% Acrylamide.. ).. for 10mL
Tris HCl (1.5M, pH 8.9) 2.5mL
SDS (10% stock soln) 0.1mL
Ammonium per sulfate (10%) 60microL
TEMED 15microL
Acrylamide:bis XmL
Add ddwater to make total volume 10mL
Stacking gel (5%).. for 5mL
Tris HCl (0.5M, pH 6.9) 1.25mL
SDS (10% stock soln) 0.05mL
Ammonium per sulfate (10%) 30microL
TEMED 8microL
Acrylamide:bis XmL
Add dd water to make total volume 5mL
Also, while pouring the gel... be very careful about the air bubbles..
couple of tricks ..1) pour gel from the sides of the gel cast (works well
if you actually take support of the glass plates while pouring the gel),
2) SDS will make bubbles when you mix the above solutions.. mix very
smoothly so as to minimize formation of air bubbles
3) when you pipette out gel to pour in the gel cast... do it smoothly.. try
not to have too many bubbles and do not pour all the gel from pipette in the
gel cast.. hold some solution in the pipette.. one more way to avoid bubbles
in gel cast while pouring the gel.
Most people use butanol saturated water or plain water to take care of the
meniscus ( for separating gel), well I use 100% propanol (for Bio-Rad mini
protean .. takes about 90 to 100 microL propanol for leveling each gel ) As
you will see.. it also takes care of bubbles which might be formed while
pouring the gel!!!
Hope these tips (and tricks) help you... and you will no more get proteins
"frowning" at you!!!
Good luck
Savita
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