semi-dry western conditions - (Mar/25/2004 )
hi all,
i'm currently being frustrated by trying to run my protein through a semi-dry blotting system.... the extracted cell protein (i'm actually looking for UCP-2, 31kDa) is eletrophoresing very weel (as demonstrated by coomassie) but i haven't managed to get it to transfer yet.
i've tried both continuous and discontinuous buffer systems, and a range of power settings (15V for 30m/1h..... 150mA for 1h.....250mA for 1h....mA per cm2 of gel for 1 h) and none of them have caused protein to show up on ponceau s staining....
i'm using sigma's semi-dry western blotter if that helps
where am i going wrong?
james
i think i've worked it out, the membrane i'm using is almost 18 months old....... the sheet that comes with it says it'll last 6 months. does this sound right?
still no joy, have a new membrane and still no transfer!!! can anyone else send me the conditions they use so i have a reference point [trying to transfer a 37kDa protein from a 12% agarose gel to a nitrocellulose membrane]
regards
james
UCP-2 is a highly basic proteins, so it should be transfered easily and quickly. check your semi-dry system to see whether other proteins (just like marker) are transfered. also check ponsinus staining of your protein, this can be done by dot the purified protein on membrane then check. if it doesn't work, you can use fast green. also, put two membranes on both sides of the gel during transfering, and stain the gel after.
good luck
Hi James
I can offer you a solution to your problem.
Give me a call.
Best regards
Andrew
07815183742
hi all,
i'm currently being frustrated by trying to run my protein through a semi-dry blotting system.... the extracted cell protein (i'm actually looking for UCP-2, 31kDa) is eletrophoresing very weel (as demonstrated by coomassie) but i haven't managed to get it to transfer yet.
i've tried both continuous and discontinuous buffer systems, and a range of power settings (15V for 30m/1h..... 150mA for 1h.....250mA for 1h....mA per cm2 of gel for 1 h) and none of them have caused protein to show up on ponceau s staining....
i'm using sigma's semi-dry western blotter if that helps
where am i going wrong?
Flashboy, what kind of membrane did you used? PVDF or Nitrocellulose?
I guess there should be no problem with the membrane even if it was 18 months old. After you found that no protein was transferred to membrane, did you coomassie stain gel for the proteins? If proteins were on the gel, I assume that you forgot to add methanol to your transfer buffer. Anyway, try this book "Protein methods 2nd edition". It may help. Good luck. 
i'm using a nitrocellulose membrane, and have now managed to partially transfer the protein by adding sds to the transfer buffer, still bot 100% though!!! am running at 5V for 90 minutes
well, bugger it i've tried a wet blotting unit and its transferred perefectly!!!! that's the last time i try semi-dry blotting!!!
that has happened to me too,somehow,I made fresh transfer buffer and it seemed to work great.what are you activating you membrane with?are u washing of the alcohol completely before putting it on the transfer unit?I usually run it at 150mA for 1.5 hours,that works best for me
hope that helps
i tried everything with the semi-dry unit, a range of voltages and ampages, different buffers and different times but never got more than 25-50% transfer. now i use a biorad wet blot unit, at 100V for 1 hour in towbin buffer, using a nitrocellulose membrane which is presoaked in towbin. get 100% transfer every time...