Western blot question- High % Gel/Low MW Protein (View forum version)



bRickboss

Posted 01 February 2013 - 01:09 PM

I'm currently trying to detect a phosphorylated-myosin light chain protein that runs around 18 kDa. I just processed a couple of blots and didn't really see much protein, leading me to believe that there might have been a problem in transfer.

I'm using an 18% polyacrylamide gel and transferred like I normally do with 10% gels (100 Volts x 60 minutes at 4 degrees), transferring to nitrocellulose. I'm thinking this is too short of a time for such a high percentage gel. Just wondering if people who had experience with high percentage gels/low MW proteins had any input regarding their protocol vs. what I'm doing. Thanks!

bob1

Posted 01 February 2013 - 02:39 PM

Actually the time is probably too long and your voltage is way too high (think how far down the gel a protein would migrate if you ran it at 100 V, now compare to the thickness of the gel...) - the protein will be blowing through the membrane. Either reduce the voltage or the time. You can also place a second membrane behind the first to attempt to catch the protein. PVDF and small pore membranes are better for binding small proteins.

PhDinAcronyms

Posted 03 February 2013 - 07:49 PM

I don't know about 18% gels but I regularly detect a 17kDa protein using 4-12% gradient gels (shows up in bottom quarter of gel), then transfer to pvdf using semi-dry transfer, 23V for 60-90min. Maybe trying a lower percentage gel would make transfer easier as 18% is fairly high.

bRickboss

Posted 04 February 2013 - 10:29 AM

Thank you to both of you. You both make very good points that I overlooked. I have some gradient gels (4-15%) and PVDF membranes that I can try using a lower voltage.

Olgitsa

Posted 08 February 2013 - 10:57 AM

Maybe trying a lower percentage gel would make transfer easier as 18% is fairly high.


I totally agree! 18% is way too thick... For detecting a 18kD protein I usually use 15% polyacrylamide gel with the same transfer condition (100V, 60min, 4oC) with no apparent problem!
Good luck! :)

shirosands

Posted 12 February 2013 - 07:36 PM

What conditions to people recommend for transfer onto Nitrocellulose membranes?

bob1

Posted 12 February 2013 - 08:15 PM

Same as for PVDF - it is protein (size) dependent and transfer type dependent.

bRickboss

Posted 18 February 2013 - 05:52 AM

Just an FYI, if anyone was wondering...I spoke to the vendor supplying the antibody and they suggested a 90 minute transfer at 70 volts that worked perfectly. A lot of the higher molecular weight markers didn't transfer, but that was to be expected. I'll switch to a lower percentage gel when I run out of the 18%s that I have.