Hello,
I performed flow cytometry using antibody Pacific Blue™ anti-mouse/rat CD29 Antibody (cat no. 102224, biolegend) in endothelial progenitor cells. Also, in the same cells I performed western blot for CD29 a.k.a integrin beta 1 using antibody from cell signalling ( Cat no. 4706). Both these antibodies are detecting the same protein, however, flow cytometry showed increased CD29 expression and western blot showed decreased. Both are very conclusive results (n=5/8 repeats). Although I think flow cytometry may pick up the signal not just from that single protein, but it could be picking up signal from protein-protein interaction (Integrin beta 1 forms dimer with integrin alpha 4-VLA-4). But I am not sure. Please guys need some insights.
Thanks a lot in advance!
The differential results you are describing implies that either one or both of your mAb reagents are not specific for CD29. While unlikely, the best way to address this issue would be to perform similar experiments using cells from a CD29 KO or knock-down in order to evaluate/confirm mAb specificity.
As this antibody detects the CD49d/CD29 heterodimer I would rather trust the flow cytometry data. In western blot this antibody may not be very reliable (and as far as I know, WB is not recommended in the data sheet). In my opinion, the analysis via flow cytometry is anyway the best way to show integrin levels because you only detect intact, properly folded, surface-exposed and thus: functional integrin beta1 levels. If you can repeat the westernblot result with an antibody that is suitable for western blot this would mean: you have less total integrin in your cell, however more (active) Integrin at the cell surface. I don't know if such a hypothesis makes sense for your project....
So, my message: trust your FC data and (optionally) try another antibody for western blots
kind regards
Hi Maulasri. Full disclosure: I believe FC is a good technique for detecting relative differences in cell populations, but it is horribly flawed if you want to use it as a quantitative (or even a semi-quantitative) biochemical technique.
First of all, you can't quantitate fluorescence. Fluorescence does not follow Beer's law (like absorbance) and is dependent on so many factors (quenching, solvent, ion concentration, fluorophore concentration, antigen, etc, etc).
Plus, data from FC are subjective because the user can determine the gating parameters that are used for any given experiment ad hoc.
So understand the limitations of using FC as a tool for doing basic biochemistry, like quantifying a target. It is just not designed to do that and I'm dismayed that so many researchers are pushing FC into areas where it definitively does not belong.
Sure, westerns are hard to quantitate, but seeing a band corresponding to your antigen is much easier to relate to and quantitate than "events" on FC.
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