WESTERN TRANSFER PROBLEM - (Oct/13/2008 )
Hi all,
I am using a invitrogen transfer system(not the iblot) to transfer proteins onto a PVDF membrane. I prewet the membrane with methanol, and then soak it in transfer buffer for a minute and use it for transfer. I was able to see the prestained markers but not the proteins with ponceau on the membrane. To confirm this , I stained the gel which I used for transfer with coomassie, to my disappointment, proteins bands were still in the gel. I used 14v constant, overnight for my transfer. 20% methanol in my transfer buffer.
Please help me out to transfer my proteins....
Thank you all.
What was your mA ? How long did you transfer? Really the whole night? I usually did this with 2,5 mA/cm² (90 mA per Mini-Gel of 8,5 cm x 5,5 cm for one gel) and for 1,5 h at 4°C. What gels are you using? Premade ones? Do you use gel-strengthener like Rhinohyde?
Do you cut your Whatman-papers to the exact size as the gel's size?
I am using a invitrogen transfer system(not the iblot) to transfer proteins onto a PVDF membrane. I prewet the membrane with methanol, and then soak it in transfer buffer for a minute and use it for transfer. I was able to see the prestained markers but not the proteins with ponceau on the membrane. To confirm this , I stained the gel which I used for transfer with coomassie, to my disappointment, proteins bands were still in the gel. I used 14v constant, overnight for my transfer. 20% methanol in my transfer buffer.
Please help me out to transfer my proteins....
Thank you all.
as Biomaus suggests, Ampere is more important than Voltage; add a little bit SDS to your transfer buffer; the filter papers should not larger than gel/membrane sandwich...
Oh, how about your filter papers, you prewet them also in transfer buffer, right? Everything is nicely wet but, right? Very important. But not too wet... 
Do you cut your Whatman-papers to the exact size as the gel's size?
The current was about 110 to 85mA. I am not using premade ones, I get the gel cassettes and I pour the gel and I don't use any gel strengtheners.
No I didnt cut the filterpaper and membrane to the exact size of my gel, will that affect the transfer?.
Anyhow i will try once with cutting everything to the exzct size.
Thanks.
how is the MW of your protein? how is its isoelectric point? if the MW is high the process of transferring is more difficult. methanol washes out sds from the protein and the only charges you can rely on for the transferring are those of your protein. in some cases higher current for shorter time may work better. you could try also to decrease the methanol proportion. moreover, if the IP is too close to the pH of the buffer (if I'm not wrong the one recommended in the invitrogen system is 7 and something) your protein might have too low charge/mass ratio to be pushed out of the gel.
O.K. the problem lies within your filterpapers. When you don't cut them to match the size of your gel, the will overlap and build up a short circuit so that the current goes from the top filter papers to the bottom filter papers without touching the gel, see. So only if everything has the same size and is neatly stacked on top the current must use the longer route through the papers on top, the gel (and membrane) and through the lower papers and to the electrode.
This is very important! Your transfer will work again if you cut everything right!