ugly merged bands in western blot - (Mar/27/2010 )

hi all-

just a quick question - how do i prevent those ugly, merged bands on film? this happens to my beta-actin band. although as internal control this might seem a pointless problem, but my postdoc is being very fussy about this! LOL

i've searched through the archive, some suggest to decrease the amount of protein loaded into each well. however, i have to load 40ug protein per well, so as to detect my another protein of interest of about 90kDa (in this new lab they routinely cut the membrane horizontally into half, the upper for the protein of interest, the lower for beta-actin). the 90kDa bands shows up nicely, but the beta-actin bands across the lanes get merged up. =s

any suggestions on solving this problem?

some details in the procedure:
- 4% stacking gel, 8% resolving gel (i'm not sure if higher percentage helps - but my current lab is very thrifty with their spending and dont think my postdoc will like the idea..)
- running at RT at 50V for ~30mins until it reaches resolving gel
- running at RT at 100V until the loading dye runs out of the gel
- transfer at 115V for 1hr in pre-chilled buffer
- primary ab (beta actin: 1:10,000 overnight at 4deg). wash 3X time TBST
- secondary ab (1:10,000) for 1hr. wash 3X with TBST
- detection via ECL plus from amersham

if i were to dilute the antibody even more, what is the dilution factor u guys usually increase? 10X? to 1:100,000 (well, that seems too diluted to me?) and usually, will u guys tackle from primary or secondary antibody?

it seems silly trying to 'perfectionized' bands for beta-actin.. still, thanks in advance for all suggestions. =)

wia84 on Mar 27 2010, 06:35 AM said:

that is why you are the tech working for the post-doc or PI.

in order to interpret changes in the levels or PTM's of a protein of interest, you need a loading control that doesn't change in response to the parameters being tested. controls are more important than you seem to think.

you can dilute the secondary antibody 2x and/or shorten the exposure.

I guess you are using the 10 well minigel? I prefer the 15wells with 1.5mm thickness. The protein will be more concentrated and better signal/noise ratio

I used to dealing with protein of 37kd, 55kd, 72kd, 95kd, 120kd, I run 7% gel and cut the membrane into tiny pieces. It was fun. LOL