For my western blot, all my target proteins are within 10-100kD observed masses. I'm concerned that the lowest is Cyt C at 12kD will transfer through and not stick to membranes with standard 0.45um pore size. I'm planning to use a TGX gel from BioRad as shown by the ladder for SDS PAGE.
Either the low molecular weight (12kD) or its relative low quantity may be the reason. You should try with the PVDF membrane with pore size 0.2um.
Hi
On the following homepage they explain very nicely how to choose whrite membrane
http://www.thermofisher.com/ch/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/transfer-proteins-western-blot/membranes-transfer-buffers-western-blotting/membranes-western-blotting.html
Good luck
Hi,
I frequently use WB to detect and quantify proteins that are 10-15kDa with a PVDF 0.45um membrane (Immobilon) and I never had a problem. I normally transfer at 400mAMP for 60 minutes. With low molecular weight proteins, you obviously need to ensure that your transfer is no longer than 1 hour (at 400mAmp). As Pawan indicated, this also depends on how much proteins you have loaded, how abundant is your protein and also how specific and sensitive is your detection antibody. But as far as the transfer is concerned, PVDF at 0.45um is not a problem with 10-15kDa proteins.
Good luck.
Hi Robert
Try with 0.2 uM pore size.
In my lab we have successfully transferred 12 KDa protein using Millipore Immobilon 0.2uM PVDF membrane.
Good luck.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques