Is there a way to quantitatively measure the relative levels of two or more proteins in a single cell line? In the linked paper the authors have used Quantitative IR-fluorescence western to do that. but its not clear to me how they have done this.
http://www.cell.com/cancer-cell/fulltext/S1535-6108(17)30364-1
Hi Sajid,
do you mean at single cell level?
- for single cell, you can use the CytoF
- for a cell line, a classic westernblot.
Good luck
Val
Hi Valentine,
How we can do it using western blot? Do you suggest incubating the membrane with two or more antibodies at the same time followed by ECL detection? If so, what we should do if the molecular weights of desired proteins are very close. Alternatively, if we probe with individual antibodies, then I believe we could not really quantify the relative levels considering different exposure times and conditions.
Thanks
Hi Sajid,
yes you can incubate the membrane with more than one antibody. in fact you always need it, to detect your protein of interest and at least your loading control protein (such as GAPDH, beta actin).
you can read some of those comments, it might be useful for you.
good luck
https://www.researchgate.net/post/Can_I_detect_two_different_proteins_in_a_western_blot_using_two_antibodies
Hi Valentine,
Thanks for elaboration.
I have never tried on simultaneously incubating loading control antibody together with antibody against protein of interest. I either cut the membrane horizontally (if they differ in molecular weights) or else I strip the membrane and then I do for loading control.
Anyway, I can try this to measure the relative levels of two different proteins in a single cell line sample..though I don't have access to Li-Cor Odyssey IR scanner right now.
You can try using a Multiplex Immune assay if you want measure many proteins of a single cell type or tissue, even works whit phosphorylated proteins and use minimum amount of sample.
I must recommend quantitative proteomics PDMD (protein-direct-microsequencing-deciphering) method instead of non-quantitative immunological method and Western blot method (please see file; HepG2 fucoidan and IEF for hydrophobic protein).
Hardships is required to achieve this new method (cell-protein determination/identification requires c.a. 1 month), however result by this quantitative method is fruitful and reliable.
I wonder about of Dr. Hayakawa's approach, as it is new for me. I agree wiht some of the responses: you cannot compare two different proteins using antibodies. Everything will turn out to be relative and not absolute. By Western or other immune-based approaches you may see trends but not calculate absolute concentration ratios.
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