Transfer Buffer - (Nov/20/2006 )
Does it affect the transfer of the protein to the membrane if methanol was not added to the tranfer buffer cause i think i used transfer buffer that did not have any methanol
The role of methanol in transfer buffer is to help good transfer of small proteins to nitrocellulose membrane and also to prevent swelling from heating and to keep proteins adsorbed to ur membrane ....
So its a must when using a nitrocellulose memebrane but if u are using PVDF membrane its ok to use transfer buffer without methanol
there are also protocols for transfer buffers without methanol; but if it has been simply forgotten, transfer will not perform optimally; nevertheless you should have transferred proteins on the blot...
I never use methanol when using pvdf membranes. works fine in my hands
The first aim of avoiding methanol was an evironmental one by reducing solvent rejections. Btw, i use to study 32 to 300 kD with nitrocelluloses membranes without any problem
methanol is used in transfer buffers to strip sds from the protein being transferred.
it is not necessary when transferring from non-denaturing gels.
Hi Guys,,,, thanks for the reply
I continued my blot. I used nitrocellulose membrane and the protein transfered to the membrane. it seems to me things were ok ( the protein i'm looking at is about 54 kDa) but im worried that the quantitation will be not aqurate enough coz there was no methanol in the tranfer buffer, do u think this will affect the quantitation of the protein ? . I think i will run the gel again and add methanol to the transfer buffer and see what will happen..... thanks
you mean quantitation by ponceau or western blot?
nothing gonna that change, but i think quantitation by these method is not really accurate. The relative ratio between rows will be kept.