I collected CM from tissue (serum free FBS), so I have very high concetration of protein, no need to concentrate.
can I just add sample buffer to the media, boil and do western?
or I am missing something?
If you have high concentration of proteins, why will you run western? You can simple detect your protein with SDS-PAGE. If you want to perform affinity binding assay, it is better to go for ELISA instead of western blotting, which is a procedure under denaturing conditions.
Thank you for your answers!
I tried do Elisa, and even after 1:200 diluation, the samples was higher than the known standarts, so I thought about doing western.
I am getting very specific and but around 150kd, when the size of my protein in 10kd. Also, i tried with 2 different antibodies from different companies, and got the exact same thing.
I cant understand what is the problem..
Is your primary Ab against your protein pure or clarified sera? what about the purity of your target protein? Is it pure? Is it from clarified cell lysate/tissue homogenate? I am speculating a non-specific binding with some contaminant proteins with a molecualr size of 150 kDa.
my protein is from conditioned media from lung (serum free), I just get rid of cells and debris using centrifugetion. and added sample buffer to the media. Do you think there is better option?
You could fractionate your sample, then run a dot-blot of fractions. Confirm protein presence by WB of positive fractions. Fractionating your sample (e.g., size exclusion) will remove many proteins that are not of interest. I always quantitate crude protein samples (i,e., BCA) as a total-loading measure/control.
- Badges
- Science method