I've been attempting to run a western blot analysis on some collagen samples and have been running into issues with the transfer process. I've gotten bands to show up on the membrane for all samples, but they're incredibly faint, even for the control.
After transferring to the membrane, I'm staining the gel and getting strong bands in all the lanes except the molecular weight, which is why I'm convinced the problem is with the transfer and not the blotting reagents. Does anyone have ideas I can try to get a better transfer?
Here's some details on what I've done/used so far:
- Test samples = milled, purified bovine tendon brought into solution with 0.01M HCl
- Control = Pepsin soluble collagen in 0.01M HCl
- 3-8% Tris-Acetate Gel
- PVDF Filter Paper Sandwich, 0.45μm Pore Size membrane
- Transferring in NuPAGE Transfer Buffer (20X) diluted with WFI water and methanol
- Transfer overnight, normally at 30mA constant current
- Tried running the transfer overnight at 100mA constant current with no significant difference
Thanks for your help!
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