When I work with this method, all proteins are not completely transferred from the gel to the membrane (PVDF), and after staining, there is still some protein remaining on the gel.
I'm looking for a way to fix this problem.
Hi,
Transferring protein from gel to membrane is a crucial step that you need to carefully optimize first because you have to know your protein size so,try and check the following(1) Your protein size and the kind of membrane you want to use(2)Transfer condition such as 100V for 30mins-2hr.(3)There are no bubbles between the gel and membrane.(4)That the membrane and gel(sandwich) are fully immersed in your transfer buffer.If you want check the quality of transfer,you can first stain with Ponceau S(0.2%(w/v) Ponceau S and 5% Glacia acetic acid).
Finally, check the amount of the protein that was not transferred.
I hope this will help you.
All the best!
Dear Sepideh,
Try this transfer buffer: 25 mM Tris Base (3.0 g/l), 195 mM glycine (14.4 g/l), 0.025% (w/v) SDS(0.25g/l) and 5% (v/v) ethanol, pH 8.30. Before transfer, incubate your gel at least 5 min in transfer buffer. Conduct semi-dry transfer for 120 min at 45 mA (for 6 x 8 cm gel) or 180 mA for bigger gel and more numbers of gels. Alternatively, use wet transfer ( 90 mA overnight at 4 C degree).
Good luck,
Mateusz
Dear Krzyscik
Do you mean that gel size affects the voltage range of the instrument? You may want to explain more or introduce an article on this topic, please.
Thank you for your guidance,
Sepideh
Hi Sepideh,
here you've got an explanation: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf
Regards,
Mateusz
Hi Krzyscik
Thank you for your guidance.
excuse me, does this measure of the voltage and time for a high-protein (120kDa)? I mean a little time (not a day or night) and high voltage.
Regards,
Sepideh
Try 350 mA/60 min or 180 mA/2.5 h, in case of the failure, 90 mA overnight.
Good luck!
Mateusz
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