I have been running several western blots over the past few days and have had the same issue where large proteins seem to stain nicely where as the smaller (~50 Kda) stain very poorly. I am using 4-12% Bis-Tris Nu-PAGE gels and loading 10-15 ug of protein into each well. I have been using 1x Tris-MOPS running buffer and running at 200 Volts. For transfer I am using a Bis-Tris Bicine transfer buffer and transferring for ~3 hours at 30 Volts. I have been blocking overnight at 4°C in BSA and TBST 0.1% SDS. For imaging I am incubating with a primary antibody, beta-actin 1/2000, DNApkcs and ATM 1/100. Finally using a secondary HRP linked antibody. I have attached 3 images, DNApkcs, ATM, and beta-actin.
Is B-actin antibody fresh?(very old/repeatedly used antibody may perform poor) and are you incubating your primary Ab for 1h at RT or overnight in cold room? later one helps if the signal is weaker; Is it dissolved in milk(I can see unspecific band nicely in wells 1,3,4..perhaps milk may help you)?.
Thanks, I have just started a new western blot and am using a synthetic blocker, SuperBlock (Thermo Scientific). I have been incubating the primary Ab for 1 hour at RT. I will see if this helps with the none specific banding. Would loading too much protein affect the b-actin band?
Ian Cartwright, for smaller mol wt. protein separation it is recommended to use 1X tris-MES running buffer. MOPS buffer is for higher molecular weight protein separation.
Also try low current + more methanol (20%-30%) while transfer for low mol.wt proteins. Hope this helps!
Perhaps this could be an issue of uneven transfer. I would trouble shoot this by looking at your protein ladder. Are the low MW markers transferring through nicely ? ; before membrane blocking, do you do some kind of protein stain (Ponseau Red) to check protein transfer ? Ponseau Red stain should be able to tell you whether proteins are been transferred evenly across all MW. If Ponseau Red stain appears even, then I would check primary and secondary antibodies.
- Badges
- Science method