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.

There is normally no dependency of the transfer times on the molecular weight. First there will happen an isotachphoresis due to the different pH in the gel and the transfer buffer. That's will take some time and the more power you are using the warmer the gels will become. It's therefor dangerous and the reason why I prefer a tank blot. Here you can cool the whole system (but not below 15 °C, SDS will precipitated below) and do the blot overnight.