I had this problem with several nitrocellulose membranes so far. After transfer I stain the membrane with Ponceau S and get some faint spots where it looks like less of the protein was transferred in these spots.
Samples are run on a 4-15% TGX Criterion gel, 180V for around 45 minutes.
The transfer is done at 2.5 A, 25V for 7 minutes to a nitrocellulose membrane.
They are not air bubbles.
It´s not a problem related to the gel (precast) or the membrane (precast) manufacture since no one else in the lab is having the same issue.
It´s not a problem related to the transfer machine or cassette, for the same reason stated previously.
It´s not a problem related to the membrane or the gel drying out since I soak them in TGX buffer briefly before transfer.
I believe it could be something related to the samples themselves.
Does anyone have any suggestion? Right now everything would be very welcome!
Dear Francesco,
If you have the problem at the same region of the membrane, I suggest you to renew the transfer buffer If you use a wet system and try an overnight transfer. Before this also check or change the fiber sponges.
Dear Ozdemir,
The membrane we use are pre-soaked. We use biorad PVDF membrane in a semi-dry transfer so I wouldn´t know how to renew the transfer buffer.
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