Western blot question- High % Gel/Low MW Protein - (Feb/01/2013 )
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!
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.
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.
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.
PhDinAcronyms on Mon Feb 4 03:49:22 2013 said:
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!
What conditions to people recommend for transfer onto Nitrocellulose membranes?
Same as for PVDF - it is protein (size) dependent and transfer type dependent.
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.