Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

non desired brigther bands - (Nov/25/2009 )

Hello all,

I'm new in western blot technique. I'm running 30microg protein content form lysis cells. I want to detect Bcl2 protein. I detect my band but also some tenue higher bands. And also a lower band which is much brigther than my target band. What I'm doing wrong? can be some degradatation of the protein? I prepare the samples by mixing with SDS and mercaptoethanol, 95C, 3 min. I don the incubation of primary antibody with 5% pbs-t overnigth at 4C and the secondary with 5% milk for 1:30h at RT. thanks a lot
Attached Image

-pba-

30 mcg is too much for a western blot!!!!!!!
we incubate with primary adn secondary for 1 hour at rt on a shaker with TBS washes 5 min 3 times each.
May be too much of protein and too long an incubation times is leading to these observations!!!
Worst case your antibody might be cross reacting!!!

-Pradeep Iyer-

OK, I'll try to reduce protein amount and incubation times. Do you use TBS or TBS-T? But for the secondary antibody I'm not able to wash completely the bands from primary antibody with 4 x 6 min washes

thanks a lot!!!!!!

-nuris-

we use TBS and give 3 washes after primary adn secondary and incubate in both antibodies for an hour after blocking for half an hour!!!
Best luck!

-Pradeep Iyer-

Titrate your antibody - do different dilutions across the same sample, this should lower the incidence of non-specific bands. Also check that the antibody you have is supposed to be for western blot - there are a lot of different ones for different applications. You may also want to play with different blocking solutions, such as BSA, or gelatin.

-bob1-

You have to consider if your protein is a low abundant protein, 30ug might just be right. I've loaded up to 50ug for WB.

-medchemgirl-

medchemgirl +1.

WBs with over 90 g is okay for some proteins. The one rule with WB is that there are no identical proteins, and that every specific Ab should be individually optimized.

I do primary Ab O/N at 4oC in 5% milk, and the second 1h at RT in 5% milk. I do 2 washes in TBS-T (for as long as you like, min 10 mins each) in between each incubation.

-madrius1-

I agree. I also do the same thing u do in reference to the incubation.

madrius1 on Dec 2 2009, 04:08 PM said:

medchemgirl +1.

WBs with over 90 g is okay for some proteins. The one rule with WB is that there are no identical proteins, and that every specific Ab should be individually optimized.

I do primary Ab O/N at 4oC in 5% milk, and the second 1h at RT in 5% milk. I do 2 washes in TBS-T (for as long as you like, min 10 mins each) in between each incubation.

-medchemgirl-