how can I take this non specific band out?? - lysis did not go well (Aug/11/2005 )
I'm having a bad luck with my western..
I'm using 2x SDS-PAGE sample loading buffer to prepare my cell lysate and this is the protocol:
10ml 10% Sodium dodecyl sulphate (SDS)
8ml 0.5M Tris pH6.8
1ml 0.1% bromophenol blue
Spin down 10^6 cells and wash in phosphate buffered saline.
Resuspend cells in 100µl warmed lysis buffer, vortex then boil for 5 - 10 minutes to break up DNA.
Cool and centrifuge to collect droplets.
Vortex briefly to mix.
following this protocol I got two bands, one of the size I'm expecting and the other band of higher molecular wieght, which to me means that the cells did not lyse will...
so my question is can I go back to the cell lysate and lyse the cells again and if so how???
I have a few suggestions and it is better you try this out in new lysates:
1. Do all steps of lysis on ice only! This is very crucial to ensure that there is no degraded proteins in your sample.
2. Why are you worried about breaking up DNA? After lysis, centrifuge till you get a clear supernatant and do a Lowry/some other appropriate protein estimation assay.
3. The higher molecular weight band could be due to a number of factors. What kind of protein are you looking at? It could be complexed with some other proteins as well. What is the specificity of your antibody?
All the Best!
Thank you for your help I'm not worried about breaking up the DNA, all I want is to clean up my lysate and having this extra band tells me that the lysis went wrong, and i was hopping to save the lyastes i have because i'm doing stimulation assay...
The protein i'm looking for is phosphokinase, the antibody reognize human p70 S6 kinase phosphorylated on Thr 412.
so what you think?
A non-specific band on a western blot does not necessarily indicate poor lysis. Based on your protocol, I would think you getting sufficient lysis. Run a sample that should not have your protein of interest and see if the higher molecular weight band shows up.
Thank you so much for your quick response and advice
I will keep you posted
I just got the results of running the control antibody actin, what you think??
I will strip the membrane and probe with my antibody taking care this time with washing may be the nonspecific bands will go away or at least have lower and lower signals...
after probing with actin, I stripped the membrane and I'm sure that there was no signal "I'm using odyssey IR system" and probed again with anti-phospho p70 S6 Kinase primary antibody, then detect with goat antirabbit IgG secondary antibody " same 2ry antibody used for actin"...
The signal I'm expecting is at 66kD, I got very strong specific signal at 42kD "actin signal "
I'm frustrated and I do not know what went wrong this time???
Please help me