I have been performing the Extracellular Flow Cytometry Procedure for detecting the extracellular protein and the data showed some reduction of protein expression (My protein is transmembrane protein). But now it turns out that the antibody I use recognize intracellular domain. So I wonder whether Extracellular procedure can also recognize intracellular epitope or my data is just the fault one? (I have repeated times, and it gave me consistent data).
Could anyone have experience give me some suggestion.
Thank you so much for all your help!
You get the data may be a background noise because it is not possible to get a positive results.
Second, you should re-compensate you data.
Or If you fixed the sample than you get this type of noise.
To be able to respond you with accurate information below information is needed:
1. Which type of cells you are dealing with?
2. When you say extracellular, do you mean a protein bound to cell surface?
3. If you have used fixation, what have you used?
4. What was your staining protocol?
If you send the information above, I may be able to help you.
Thank you so much for all your suggestion.
# Dear Dr. Hari, Thank you so much for your suggestion, but what make me confused is that there is some reduction in the treated group compared with control group. So this may not be just due to background noise. Is there any problem with the non-specificity of antibody I used (Eventhough, it is monoclonal antibody).
# Dear Ms. Gulderen, for the information needed:
Above is information about my protocol, I hope you could give me some advice.
Thank you so much.
I suggest that you validate your centrifugation speed and period used for repeated washings first. I assume that PBS is fresh (daily prepared) and have a pH of 7.2-7.4. Then to make your protocol finer, you need to validate each and every step. With monocytes, it is easy to have membrane permeabilisation during centrifugation. Also, some MoAb's do stain both surface and intracellular compounds. Have you titrated your secondary antibody, if not, that could be a problem also.
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