western blot trouble shoot-plz help - (Apr/20/2005 )
Hi All,
I have a strange problem with western blots. AFter the transfer of the gel on to a nitrocellulose membrane we use a reagent (Ponceau solution ) to check the transfer.
THis time when i used the reagent, it showed that only the top part of the gel was transferred and not the bottom part. 
This reagent (ponceau) , all it does is stain all the bands.
Has anyone seen anything like this ?
plz help me out 
it it possible that you had an air bubble in your transfer sandwich - thats happened to me and i've had big blank patches on my membrane. What standards did you use?
Jon 
i am afraid that your PONCEAU SOLUTION expired.
Jon
thanks for the replies.
At first even i thought there must be something wrong with my ponceau solution....but if ponceau is bad then it shudnt show any bands at all. It showed bands on the top part and not in the bottom part.
Coming to air bubble, i have seen air bubbles earlier in my blots but they where small and round. this looks like a square blank with no bands.
anymore suggestions wud be really helpful
Are using liquid or semidry transfer appartus? which half of the blot that deos not stain with the Pon stain is it the low or high molecular weight?
Tow possibilities:
1- The current is not flowing on half of the gel, so you need to check your transfer appratus.
2- You have transfer time was short so the high molecular weight proteins > 50 kDA did not transfer appropriatly. Or your transfer time was lengthy and low molecualr weight proteins has ran through the membrane!!!!
I hope this is helpful, Good Luck111 
Try using a prestained protein molecular weight marker-it should transfer in color onto your membrane so there is no question of orientation and usually a good marker transfer equates to good protein transfer throughout the gel unless you have bubbles.
Tow possibilities:
1- The current is not flowing on half of the gel, so you need to check your transfer appratus.
2- You have transfer time was short so the high molecular weight proteins > 50 kDA did not transfer appropriatly. Or your transfer time was lengthy and low molecualr weight proteins has ran through the membrane!!!!
I hope this is helpful, Good Luck111