7 replies to this topic

[pba]

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, 95ºC, 3 min. I don the incubation of primary antibody with 5% pbs-t overnigth at 4ºC and the secondary with 5% milk for 1:30h at RT. thanks a lot

[Prep!]

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!!!

[nuris]

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!!!!!!

[Prep!]

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!

[bob1]

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.

[medchemgirl]

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.

[madrius1]

medchemgirl +1.

WBs with over 90 µg 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]

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 µg 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.