Does it have to have HRP link in particular?
Can it have FITC link or I can use unconjugated primary antibody?
Please see this research gate link
https://www.researchgate.net/post/Can_I_use_an_HRP_conjugated_primary_antibody_for_WB_with_no_secondary
Dear Biraj,
You want probably to make Western Blotting or at least a reaction with chemiluminescent detection. For the primary antibody to use, what is important is not the coupling to anything but the species you are using. In fact this is like for immunofluorescence (IF) that you probably already did or at least red. You usually use a first unconjugated primary antibody designed against your protein of interest and then a secondary that has to be affinity purified against the species of your primary antibody (most frequently anti-mouse or anti rabbit IgG) and this is this antibody that is conjugated to horseradish peroxidase(HRP) for chemiluminescent detection.
Hope it will help. Good luck for your experiments.
Veronique Proux-Gillardeaux :
Thank you so much, Veronique. I have primary antibodies raised in rabbit against the protein of interest. The secondary antibody that I use is IgG from rabbit and is HRP linked.
I am in the process of purchasing antibodies (primary) against the periplasmic proteins of Pseudomonas aeruginosa. The vendor I am looking at carries such an antibody raised in rabbit and with these conjugations: Biotin, HRP ad FITC.
I realize that it is the secondary antibody that is involved in the chemiluminescent detection. I was curious to know if having a FITC tag in the primary antibody affects the secondary antibody binding or the HRP associated chemiluminescent detection in any way.
Dear Biraj
I never tested to use a primary antibody coupled to FITC and a secondary to HRP, it could work but it could also impair the recognition. If you will only use it for HRP experiments why not using the already coupled to HRP? You will have less nonspecific signals (but also no amplification so perhaps less signal) or prefer the not conjugated primary antibody for all types of experiments.
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