If i need to do western blot of two proteins of varying sizes separated in the same gel (one 55kDa and one 13kDa), what should be the membrane transfer conditions that I need to use? Is 70V for 2 hours good?
Run the proteins in 10-12% SDS PAGE gel with 15Amp current till the dye crosses stacking gel and then run with 45Amp till end. Membrane transfer conditions varies according to gel sizes. i.e 0.8 mAmp per cm2 of your PVDF membrane if you are using a Semi-Dry Transfer Apparatus .
Hi Prabhadevi,
I routinely transfer both large (160 kDa) and small (17 kDa) proteins without any (known) problems. We use a system from Invitrogen (http://www.lifetechnologies.com/order/catalog/product/EI9051?ICID=cvc-Western-Blot-Transfer-c1t1) for transfer, and our standard setting is to transfer for 1h at 35 volts, variable amperage. You may put two membranes in the transfer sandwich to control for transferring through the first membrane, especially for your small protein.
Good luck with the experiment!
Hello Prabhadevi,
I usually transfer different protein sizes ( 11- 75 KDa) in one gel with this condition ( 250 mA, 1hr, RT), and it works very well with both type of membranes PVDF and Nitrocellulose.
Good luck
Hi all,ly
Thank you very much for your valuable suggestions. I will try out my experiment by incorporating your suggestions in my protocol.
Good to take higher percentage or gradient ready gel and transfer different protein sizes ( 11- 75 KDa) on membrane. For transfer use nitroculose or PVDF membrane at room temperature applying condition -250 mA for 1 hr.
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