Protein bands on the bottom of the gel - (Mar/02/2010 )

I run electrophoresis while the day does not come until the end of the gel. After gel staining, no protein low Mw and proteins high Mw are in the middle of the gel (instead of above). I used 12 and 15% gel on which, according to the protocols, all the proteins shoud be separated. Is there any idea where I made a mistake?

you may have overrun your gel.

are you using a tracking dye (usually bromphenol blue)?

you should let it migrate to the bottom of the gel then stop the gel and stain.

mdfenko on Mar 2 2010, 10:12 PM said:

Yes, I am. When tracking dye migrated to the bottom of the gel I stoped the run and stained gel but there were not low Mw proteins . This is A-Page and I use methyl green

a-page?

maybe the gel percentage is wrong?

is the crosslinker ratio correct?

percentage and crosslinker may be off if the stock is prepared incorrectly.

was the gel completely polymerized?

have you had success in the past?

are any of the solutions newly prepared since the last successful run?

can you show us a picture?

What size are the low MW proteins?
Are you using a stacking gel?
Has this worked for you before?

There are many steps at which this could be going wrong -- a picture would be very helpful.

What size gel are you using? What voltage do you run the gel at? Is your power supply set for constant current? How long does the gel usually run before the tracking dye reached the bottom of the gel?

What is your running buffer recipe? What is your tracking dye recipe?

Is the gel commercially prepared, or do you cast them yourself? If you cast them yourself, what are your recipes for the stacking and separating gels? Is your ammonium persulfate and TEMED freshly made? Do you use an overlay? How long are you allowing the gel to polymerize?

Are the low molecular weight bands of your molecular weight ladder still visible on the gel?

What is your sample? Are you sure the low molecular weight proteins you're looking for are actually in your sample?