Memrane for Western Blot- which are the best? - (Nov/15/2006 )
H!
I'm new in Western Blot , but would like to know your opinion about the membranes you are using. Which company, type, etc.. Thank you for help.
H!I'm new in Western Blot , but would like to know your opinion about the membranes you are using. Which company, type, etc.. Thank you for help.
Generally, transferring large proteins such as antigens, Ig, glycoreceptors, histones, etc works fine on 0.45 micrometer nitrocellulose membranes, while low mol weight proteins or nucleic acids are bettter transferred on smaller pore size paper (0.2).
PVDF membranes are chemically resistant and recommended for their higher protein binding capacity and resistance to tearing, allowing stripping and reprobing. There are PVDFs for western blotting (chemi and colorimetric blots), and PVDFs specifically designed for protein sequencing. I think the company is not that crucial as long as it's a recognized brand. I'm currently using Bio-Rad's membranes.
More important is the equipment that you plan to use for transferring proteins - it very much depends on the size of the proteins to be transferred, and on the homogeneity of your samples (if a mixture of different protein sizes or a prefractionated sample).
lillymay
I agree with Lillymay.
I use membranes Immobilon from Millipore and they work really well, I recommend them. They offer you a wide range of types depending on your application. In addition, membranes are sold both in sheets of different sizes and rolls. I buy a roll and cut into the size I need, it´s easy and cheaper.
Good luck
I use membranes Immobilon from Millipore and they work really well, I recommend them. They offer you a wide range of types depending on your application. In addition, membranes are sold both in sheets of different sizes and rolls. I buy a roll and cut into the size I need, it´s easy and cheaper.
Good luck
Thank you, I will then try with Immobilon. My protein is 90kDa size, and I'm planing to use semi-dry system for transfer.
My protein is 50 KDa. Some time ago I used wet transfer but now I do semidry and is really better!! I transfer for 30 min at 15 V using the TransBlot Semidry Electrophoretic Transfer Cell from BioRad. It saves you a lot of time, work and buffer. In this link there is a nice protocol:
TransBlot
If you choose PVDF membranes (which I recommend) don´t forget to activate them in methanol for a few seconds prior to transfer.
TransBlot
If you choose PVDF membranes (which I recommend) don´t forget to activate them in methanol for a few seconds prior to transfer.
I also encourage using TransBlot SD for such proteins. I agree with Veteran Pumuki, it's really comfortable and money saving. But if you don't have it, go for wet transfer, it works too. Never try SD transfers on large proteins (multimeric). The short transfer time will lead to bad results.