I want to biotinylate polyclonal antibodies. The application of these polyclonal antibodies would be in the lateral flow assay. I am not looking for any commercial kit. Please help with the accurate reproducible protocols
Hi Snehal sangram Jamalpure
You can refer to the protocol given below.
https://www.drmr.com/abcon/Biotin.html
Good Luck.
The protocol by DRMR in my opinion is highly misleading. Biotin itself, as the reagent is called in the protocol, won't work at all, it needs to be activated, e.g. as NHS-Ester. These active esters are available commercially. Look for NHS-Biotin or Biotin-X-OSU.
If you are not familiar with biotinylation i would strongly advise you to use a kit to get started, as its a false economy if you have to purchase and make up all the suggested reagents.
However, if you want to use the above protocol, please note that it is very ambiguous. At no point in the process will you ever use "biotin" despite what the protocol says. The biotin reagent that it refers to is not explicitly stated, but when biotin is mentioned it really means "biotin-NHS ester". The protocol alludes to alternative products with longer linkers but with no guidance on what to use. The safest single option is to use biotin-LC-NHS ester. However, once you've added up all the costs of the reagents you will surely find that a complete kit is cheaper.
Re Kit: Depends what you already have in the lab. Roche Diagnostics e.g. is offering a set that includes NHS-X-Biotin (X is a C6 spacer, I second Nick's recommendation, otherwise the biotinylated protein frequently won't reach into the binding pockets of streptavidin) plus PD-10 columns and some DMSO. The DMSO should be fresh, but I would not panic about having it absolutely anhydrous (as DRMR states). You will dump the reagent into an aqueous solution of your protein anyway). Just prepare a minimal amount (possibly limited by what you can weigh reliably) of a fresh solution every time and use it immediately. You might need to play a little with the ratio of NHS-Biotin to protein for optimal results.
It might make sense to aliquot the DMSO and to store it and also the NHS-ester tightly sealed in a freezer. Before you open the vials, let them warm up to room temperature to avoid condensation of water.
And, most important avoid buffers with amines (e.g. Tris) for the coupling reaction. They will compete with your protein for the reagent and thus scavenge it.
Thank you Nick Gee and Wolfgang Schechinger for your response. I now understood the nuances of the biotinylation protocol.
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