Hi,
I ran SDS-PAGE followed by a wet transfer into a PVDF membrane, but I did not get a good transfer.
For cell lysis, I used RIPA buffer. I used Fluorescent Compatible Sample Buffer (4X) and DTT to reduce the samples. I heated the samples for 10 minutes at 70 ˚C.
For the electrophoresis, I used a Tris-Gly 4%-20% gel and ran the samples for 90 minutes at 125 constant V. I had 30 µg per well.
For the wet transfer, I used Tris-Gly transfer buffer with 20% methanol at a constant V of 25 for 2 hours.
After transfer, I used the no-staining labeling reagents from thermofisher to check transfer efficiency.
As you can see in the image, most of the protein stayed in the gel after the transfer, and the middle lanes became narrow. The ladder transferred well.
Does anybody have this problem before?
Thank you
This might be due to DNA contamination, genomic DNA in the cell lysate may cause the sample to become viscous, resulting in protein aggregation.
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/removal_dna/
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-gel-electrophoresis-information/western-blot-troubleshooting.html
Hi Alfredo Reyes Oliveras
Your main issue is the transfer. 25 V for 2 hours is not enough voltage of time. If you want to do a quick incubation, it should be 90 V for 2 hours (but remeber to place in the fridge to cool down the buffer as this can affect the transfer! Alternatively, you could run the gel for 720 minutes at 30 V.
Either of these two methods should remove all proteins from the gel.
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