Hey Guys,
I performed a western at 90 volts for 1 hr 30min, transfer using iblot dry transfer system to a PVDF membrane, 1 hour block in 5% milk and incubated with primary overnight in BSA and secondary in milk for 1hr at room temp. washes 3x in TBST20.
When I came to image, I see dark smears accross where my bands of interest are (15kDa), when inverted the smear turns white, I have attached am image of my blot,
any ideas as to the cause and how to possibly rectify this?
Thank you very much for any guidance
Sounds to me like uneven transfer, or the membrane not covered/stuck to the walls of the vessel while incubating with the antibodies.
Dry transfers are sometimes tricky, do you have a "submarine" transfer cell?
Thank you Alejandro, I will try stripping the membrane and re-probing again. Unfortunately we do no have any 'submarine' transfer cells in our lab
Thank you for your response :)
This could be a transfer problem as Alejandro said, wet transfere can give better results in this case. Also this could be happened due to high or low antibody conc. Mind the voltage as well when doing transfer step because high voltage increase the temp and may break down your protein. Putting the tank on ice while transfer may reduce the temp. Regards
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