eNOS ELISA - (Jul/16/2008 )
I must design my own eNOS ELISA, the commercial kits are expensive, and I have hundreds of samples.
I checked one Sigma mouse anti-eNOS antibody with anti mouse IgG-Peroxidase conjugate and it works (my conjugate detects anti-eNos antibody coated on ELISA plate). But still I need capture antibody. Can you recommend me any antibody which can be used as capture antibody? Or antibody pair suitable in my ELISA? I was ythinking about R&D Systems AF950 antibody, but maybe someone has any experience in eNOS ELISA and can recommend me something else?
I've been investigating the upregulation of enos within lipid rafts. I use:
BD Biosciences Mouse anti-eNOS (Human, mouse, rat) Catalog # 610297
It works for me, although I do not use elisa... however this antibody has been tested and works for Immunofluorescence, Immunohistochemistry, Immunoprecipitation, and Western Blotting. It should work well for an elisa...
I do wonder why you've chosen to use ELISA... If you have access to a flow cytometer, I strongly recommend you go with flow cytometry...
With an elisa, you measure the over all concentration in a batch of cells...
If you use flow cytometry, you measure the SPECIFIC concentration of eNOS in an individual cell, but you do this for 5000 to 1,000,000 (or more) cells...
SOOOOO, with flow cytometry you will get a specific protein (eNOS) density per cell. Since you'll be doing thousands to millions of cells that density (micrograms/cell OR protein/cell) will come with a nice statistical analysis giving you the mean value, and standard deviation.
This being the case, if you want to subject to cell to some experimental condition, you can then measure the eNOS concentration and you'll be able to see the increase or decrease in cell density and publish it with statistical certainty.
Thanks for response.
I've used this antibody previously in WB, but I couldn’t get any positive signal. Later I tested this antibody in sandwich ELISA without success.
The idea with flow cytometry seems to be ok, can you send me please a detailed protocol? I will try it.
Thanks a lot.