I'm attempting to detect a protein of interest (NQO1) via western blot for OVCAR-8 cells, however, while the actin levels are consistent the protein itself does not appear on the gel (chemiluminesent detection). I've used between 15-30 ug samples, which is sufficient for the other ovarian cancer cell lines I'm using to detect the same protein. Interestingly, I can use the same primary antibody and am able detect it with indirect flow cytometry, and immunocytochemistry using a secondary conjugated-fluorophore. While the fluorescence intensities are low, it's still being detected. I also ran auto fluorescent controls in for the flow cytometry, as well as samples where only the secondary conjugated-fluorophore was used, and there was not a significant difference between the two controls, so I assumed the signal from the sample to be accurate. Is there another explanation for why it's not appearing on the western blot? Should I increase the sample concentration? Also, NQO1 activity studies also reveal that it is active, although its activity is lower than what I'm seeing with another ovarian cell line (OVCAR-5).
I believe that the antibody that you are using is a monoclonal antibody that recognizes the native form of the target protein.
Western blotting denatures the protein, thereby the natural epitopes are destroyed.
You may try a polyclonal antibody for the selected protein if commercially available, also check if the tech sheet if it supports western blotting.
I believe that the antibody that you are using is a monoclonal antibody that recognizes the native form of the target protein.
Western blotting denatures the protein, thereby the natural epitopes are destroyed.
You may try a polyclonal antibody for the selected protein if commercially available, also check if the tech sheet if it supports western blotting.
Sudeep Kumar Hua Xiao thank you! According to the supplier it is suitable for western blots, and I’ve used it with different cell lines for westerns and it works fine. Also, I’ve taken the same cell line (OVCAR-8) and generated spheroids and was able to detect the protein using the same antibody via western blotting, but in monolayer culture there’s nothing on the gel.
Your last description of results is curious. So overall what I have gathered is that you are using a commercial antibody that is for both flow and westerns and in one culture condition you can detect by western and another you cannot; but you can detect by flow in both? Is this correct? You also seem to suggest that the exact reagents you are using can be successfully used to extract protein from other monolayer cultures. Correct?
William Haynes I only use flow in the monolayers, but the rest is correct.
Hi Milcah,
I can think of several possibilities that can occur on your protein of interest in the monolayer culture compared with spheroid:
1. Your protein is not expressing in the monolayer form or undergoing protein degradation machinery.
2. The alternative splicing is affected in a way that changes the epitope.
3. The protein undergoes SDS-resistant thiol-independent aggregation, which prevents the migration through the gel or conceal the epitope.
To find out the reason, I suggest, first doing a transcriptome study by comparing two forms of culturing, to see if the expression level is different. Second, you can do dot-blot to find out if the antibody can directly recognize your protein in the native state. You can also examine different antibodies for the target protein if available.
Good luck
Assuming that you are using a primary+secondary staining for flow cytometry, are you sure that the signal is specific? Have you done a proper titration of both primary and secondary antibodies? I would also confirm expression by PCR to exclude any artifact in flow cytometry.
You can increase the concentration of the protein lysate and also try to lower the antibody dilution.
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