I have biotinylated my antibody using "EZ-Link Sulfo-NHS-LC-LC-Biotin" and I'm hoping to detect it using streptavidin-phycoerthrin substrates.
I know that PE Biotin has major excitation peaks at 498 and 565 nm and minor ones at 380nm, 310nm. & 280 nm. A major emission peak is at 576 nm and minor peak is at 490 nm. I'm using a Gel Doc machine from Biorad to detect my western blot using the standard filter which has a range between 535-640 nm. Sadly I'm unable to see anything. Is there another way to check for the success of biotinylation?
-----UPDATE------
- Although using HABA would be the best method, I do not have access to it. I need to figure the success of my biotinylation within this week & it would take at least few weeks it to be shipped if I purchase it tomorrow. Is there another method of detection?
You could use HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) which is a reagent that enables quick estimation of the mole-to-mole ratio of biotin to protein in a solution. There is a kit available from Pierce/ thermo scientific for the same. Or you could just spot your biotinylated antibody antibody on a nitorocellulose membrane and visualize using Streptavidin-enzyme conjugate and colorimetric substrate. Another option is to do the same in ELISA well.
You could use HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) which is a reagent that enables quick estimation of the mole-to-mole ratio of biotin to protein in a solution. There is a kit available from Pierce/ thermo scientific for the same. Or you could just spot your biotinylated antibody antibody on a nitorocellulose membrane and visualize using Streptavidin-enzyme conjugate and colorimetric substrate. Another option is to do the same in ELISA well.
Yes, HABA assay is very easy way to test this . Add approximately 50/500 mole excess of HABA to your streptavidin and incubate for 30 minutes at room temperature, this will give an absorbance at around 500nm in UV-Vis resulting from HABA bound to SA. If you add your biotinylated antibody to this solution in stoichiometric amount, this will cause decrease in absorbance at 500nm reflecting successful binding of biotin to streptavidin replacing HABA. ( Also it might be that your Biotin labeling was a success but western blot transfer was not)
Thank you Anindya. The problem is that I have to know whether
my biotinylation worked in the next few days. We currently do not have HABA in our lab and it would take at least 3 weeks for it to be shipped. Is there another way?
Thats a bummer, You can give it a try with immunoprecipitation of your biotinylated antibody with streptavidin and then run on a denaturing gel to see whether your antibody is there are not. I am not sure though whether thats gonna work. Another quick and dirty method is MALDI mass spectroscopy ,if you have access to it. You can probably see the mass difference of biotinylated and unmodified antibody.
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