Hello everyone,
I practiced protein extraction for my non-senescent and senescent HDF samples and performed western blot.
My results show spotty bands for caveolin1. My senior says that I got some problem with BCA assay, which I cannot figure out and have to solve it myself instead of asking him again. It's kinda test for me. Each internet source has its own trouble shooting reasons which makes me even more confused. I am attaching my results and calculations for reference. I would love to hear some valuable suggestions from you guys.
Thank you
*would really appreciate if someone can figure out problem in protein assay step
Shahbaz, i used this kit https://www.thermofisher.com/order/catalog/product/23225
and linear working range for BSA equals 20 to 2000 µg/mL. Aslo you should compatibility table for buffer https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
I had some problem ( spotty bands ) for PAGE SDS, you need wait more time ( as min 30-60 min) for polymerization acrylamide and follow pharmacopoeia
I agree to the beforementioned comments/protocol.
Alternatively, spots on blots may be a result of non-optimal blocking (reagent, solvent, dilution) or of Ab quality (lot, mix etc.).
is there some problem in my sample making? Could it be a reason that I used enough lysis buffer for non-senescent cells, while very less for old cells?
Katerina Kouzari I used 2ug of each sample and 100ug of BCA reagents under 50:1, incubated for 20min and measured OD at 562nm
Sure, it may be possible that the volume may have direct influence on the generated sample solution.
Hi , primarily i would have agreed with @Andreas that the antibody binding , was a result of non optimal blocking. That can be fixed with extra reagent such as femto. But knowing the fact that caveolin-1 it is highly expressed in non-senescent cells ill agree this might be a problem with volume .
Theres a possibility the antibody you have bought might not be as sensitive as you would like if its from a company you haven
* tested it before. What Antonina recommended (thermofischer kit) is a good option. I think some people also uses Sigma Aldrich's.
In parts I agree to Katerinas comments. However, I don`t think that additional reagents are needed. In my opinion, a simple revision of the protocol and method as well as a more uniform sample treatment/preparation would help to optimize the outcome and reduce uncertainties associated to methodological issues.
Katerina Kouzari Andreas Eisenreich My past results make it certain that my antibody got no issue, but some problem in my extraction or assay process.
As I mentioned before, it is a task given by my senior and I have to do something with my current protein samples to get good results. Is it possible that I re-perform BCA assay and set volumes by taking ratio for OD values?
Are you sure of your protein concentrations? Several hundred mg/ml is unusual.
I agree to Engelberts response as well as your suggestion Shahbaz I would propose to repeat concentration measurement via BCA or alternative methods. Maybe, an alternative to a Bradford method. Possibly, measurement following methods of Lowry independent of detergences in your sample would maybe led to more reliable results regarding protein concentration.
i agreed eith Dr. Engelbrt that values are unusual, and ill agree with Dr. Andreas that another protein assay should be done. Ill suggest a modified BCA such us Micro BCA Protein Assay, in order to eliminate assay reduction (inhibition) or lowry as forementioned.
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