Protein Isolation and Gel Electrophoresis:
- Total protein from mammalian cells was isolated using RIPA buffer following the manufacturer’s protocol.
- The proteins were then run on a 3-8% Tris-Acetate Gradient Gel at 150 V for 2 hours using an Invitrogen Mini Gel tank in a cold room 4°C.
Transfer Conditions:
- The transfer was performed at 4°C using the Invitrogen mini blot module. Given that our protein of interest is a high-molecular-weight protein (~500 kDa), we followed the wet transfer conditions recommended by the manufacturer and ran at 27 V for 3 hours.
- PVDF Membrane was blocked with blocking solution (3% BSA in 1x TBS + 0.05% Tween-20) for 1 hr at room temperature.
- Further, after washing 3 times in TBST for 5 min, primary antibody was incubated overnight at 4°C with gentle shaking.
- After the membrane was washed 3 times with TBST for 5-10 minutes each, it was kept at room temperature for 1 hour for secondary antibody incubation.
Could anyone please provide any insights or recommendations for addressing this problem? Additionally, if there are any troubleshooting steps or alternative products that you would suggest, I would be eager to hear your suggestions.
Dear Dr. Yugantak Raj Gupta
I would like to suggest the following.
1. The composition of your transfer buffer is critical when you are detecting high molecular weight proteins. Large proteins can precipitate out in the presence of methanol. Avoid this by decreasing the methanol percentage (10% or less) in your transfer buffer.
2. Large proteins can precipitate in the gel, inhibiting their transfer. SDS added during SDS-PAGE usually takes care of this problem, but larger proteins might require some more SDS. So, you may add up to 0.1% SDS in your transfer buffer to discourage precipitation. However, SDS can inhibit binding of proteins to membranes. So, try starting with a lower concentration of SDS (for instance, 0.0375%).
3. For large molecular weight proteins, you have used PVDF membrane for transfer which is right. Large proteins can precipitate out in the presence of methanol. PVDF membranes do not require any methanol in the transfer buffer. So, you have a higher chance of successfully transferring your protein to the blot using these membranes. Just don’t forget to activate the membrane with methanol before using it.
4. For high molecular weight proteins, electrophoretic transfer should be done using wet transfer conditions, which you have carried out. Wet conditions are usually more reliable as it is less likely to dry out the gel and is preferred for larger proteins.
5. Getting a good transfer efficiency with large proteins might take a little bit more time. It can be hard to move those big molecules around quickly. Instead of running at 27 V for 3 hours, try transferring overnight at 4 degree C at 40mA, keeping your transfer cool.
The rest of the steps seems okay. I hope the primary and secondary antibody concentrations have been optimized.
Good Luck!
Best Wishes,
Malcolm Nobre
Dear Malcolm Nobre
Thanks for taking the time to respond. I don't use methanol in my transfer buffer, but I do activate the PVDF membrane in methanol. I will incorporate your suggestion into my next experiment.
Yugantak Raj Gupta I'm also interested in transfering to PVDF membrane a large MW protein (560 KDa) from whole tissue extracts with the Mini Blot Module from Thermo Fisher. Could you share with me your final protocol (if you were able to get reliable and consistent results?).
Thank you very much!
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