Hello. I need to detect a 100 KDa protein.
I loaded 40 ug of samples in gel polyacrylamide 8%., SDS page in the mini gel-Biorad system, wet transfer (+20% methanol), 300 mA 1h. Nitrocellulose membrane 0.45 Um. The Transfer seems to be ok for the ladder. I stained the gel with Coomassie Blue and no proteins are there. However, both the Ponceau Red Staining (10 min) and the western blotting detection give no band at 100 kDa.
Attached is the photo of the membrane stained with Ponceau.
Any suggestions?
Thanks
Please this is an electropherogram, before answering your question I want to know which gel you used (agarose gel?)
Ladders are optimized for efficient transfer. Looking at the membrane, it seems like the high molecular weight protein (100kDa) in your case has not been transferred. You can try o/n wet transfer at 30V in cold/4degree.
The low molecular weight proteins have transferred, however, the high molecular weights haven't. Dr. Pathak has already suggested a 30V wet transfer overnight. You may try and perform the transfer (wet) at 100V for 90-120mins at 4°C. This will help the larger mol. wt. proteins to get transferred onto the membrane.
When transferring large proteins, methanol is counter-indicated (it aids binding of small proteins to the membrane, but prevents the mobilisation of large proteins from the gel). In general, I have had much better success using Dunn's (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3, with 50 µM SDS for large proteins) rather than Towbin's buffer, and tank rather than semi-dry blotting apparatus (better heat distribution). Also, PVDF membranes work better than NC, as they have a higher protein binding capacity and affinity.
Thank you all. I will set up the transfer with your recommendations and let you know.
To answer Dr. Foued Zouai, I used a Tris-glycine polyacrylamide gel 8%.
For large proteins, the addition of 0.1% SDS to the western blot transfer buffer can increase the efficiency of transfer.
You can use:
For Transfer Buffer (1x) 500 ml
50 ml of 10x SDS-PAGE running buffer +100 ml of Methanol (final 20% methanol) + 350 ml ddH2O
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes