Incomplete transfer - (Aug/01/2012 )
I have a problem with the protein transfer. Although I can see the bands on the membrane after Ponceau staining, there are still a lot of bands of different molecular weights in the gel when I stain it with Coomasie. How can I improve the transfer? Can one still hope that the samples are transered equaly and proceed with antibody detection?
I use wet blotting in BioRad MiniProtean chamber. The buffer is 25 mM Tris, 192 mM Glycin and 25% Methanol. The membrane is nitrocellulose. I transfer at 350 mA for 1 h
Do I have too much methanol (I see 20% on most protocols)?
Do I need SDS in the buffer?
And should I better switch to constant voltage?
Thanks a lot!
methanol should be 10-20%.
you can add up to 0.05% sds (with 20% methanol) to facilitate transfer (good for use with high molecular weight proteins).
you can transfer longer.
what pore size are you using? 0.2um is good (less blow-through) for low as well as high molecular weight proteins.
I am going to try the buffer with 20% methanol and SDS, thanks!
we use the membrane with pore size 0.45 µm. do you think I shoud switch to 0.2?
we routinely use 0.2um membranes. less blow-through than with 0.45um especially with longer transfer times and/or low molecular weight proteins and peptides.
the choice is yours.
Which type of transfer do you use? I asume you use wet transfer. I usually use TG plus 20% methanol, 2 hours at about 110-130V (on average, the current is about 350-400mA) and keep the transfer tank cold by putting it into a box containing ice.
Do you feel your buffer is over-heated after transfer? I know of colleges reporting transfer issues and the solved when they avoided the over-heating...
I also let both membrane and gel in transfer buffer for at least 15 minutes to equilibrate them... that also help to eliminate the SDS from the gel (coming from the electrophoresis buffer)
Methanol concentration can be lower: 10 to 20%. Specially, if you use PDVF membranes 10% is usually enough. I also found PDVF membranes work better (on my hands) than nitrocellulose, specialy if you save and reprobe them. However, I have used nitrocellulose in your pore size (0,45um) and obtained good results. Another option if you do not want to change membrane composition is change pore size as mdfenko suggested.
Concerning adding SDS, that might help, just do not exceed 0.05% as recommended by mdfenko... this may be useful if you work with membrane proteins...
we use wet transfer. we put a block of ice into the chamber, but after 1 h of transfer the buffer gets warm. maybe I can put the whole chamber in a box with ice as you suggest. I will also try different membranes