Same Non-specific bands show up for different Abs!! - (Jul/27/2011 )

Hi Everyone,
I am facing a real problem with my WB. It happened that I have different antibodies that I am working with and they all have different sizes (37, 50, 55,65, 95, 100, and 117 KD), so the % of the gel and the transfer time varies. Lately, I have been seen very strong nonspecific bands that show up around 50KD and 25KD (strong enough to see light brown bands on the membrane by the naked eye after developing the membrane). Those bands appeared in all the samples and almost for every WB i ran. I have used different buffers, different concentration of the primary Ab's (1:100- 1:3000) as well the secondary.

I looked online if other people seen similar problem, and some suggest that it is the heavy and light chain of the ab. However, they seem to get these non-specific bands only when they do Immunoprecipitation. It wont be such a big problem if I see the bands for my protein, but those protein are nowhere to be seen on the blot (if not overlapping with my target protein of similar size).

My question(s) here is where did these bands came from and how to get ride of them? Why do I see them over and over again for different ab's? and does this have anything to do with me not seeing bands for my proteins ???

SEE attached pic for a sample of the bands


PLEASE HELPPPPPPP !!!!

What exactly are you doing? Please supply protocols so that we can help you to trouble shoot.

bob1 on Thu Jul 28 10:20:01 2011 said:

Hi Bob,
I follow very standard procedure for WB.
I prepare all of my buffers and gel here in the lab

Here is what I do.

Lyse the cell in 1xRIPA+PMSF and Protease inhibitor, incubate at 4C for 30 min
spin samples and collect supernatant. Add 5x Laemmli buffer +5% 2-ME, boil samples at 100 5 min
migrate samples on 12.5 % or 8% (for small or large proteins) for 1hr at 60 volts then 100v for another hr
Activate PVDF membrane with methanol for 15 sec
Transfer to membrane in transfer buffer with 20% methanol (I add 0.05% SDS for large protein).
Transfer done at 100 volts for 1.5 hr for small proteins, or 30 volts O/N for large proteins at 4C.
Block 1hr with 5%BSA in TBST (0.05% tween)
add primary Ab O/N at 4C (or 2 hr at RT)
wash with TBST
Add secondary HRP conjugated for 1 hr at RT
wash with TBST
add BIO-Rad substrate for 5 min RT ..dark
Use chem-dock for visualizing



Thanks for your help in advance

could be artifacts caused by aging b-me (or dtt) and keratins from dust. they will stain non-specifically.

SO you aren't doing IPs then? Otherwise I am out of suggestions, though mdfenko's idea seems like a good one.

mdfenko on Thu Jul 28 20:57:36 2011 said:

I add the b-me to the lammille buffer right before use, unless the stock solution itself has gone bad

bob1 on Fri Jul 29 00:09:38 2011 said:

We see such bands in samples that have gone through IP and non-IP process. We figured that during the IP process the antibody and the agarose beads need to be crossed linked before precipitating, (or by using an antibody for WB that was raised on a different animal from those we used for IP) but since we see such bands in non-IP samples we couldnt figure out whats causing this.


Thanks to you all

HELP !!! on Fri Jul 29 04:36:28 2011 said:

the b-me stock will age.