Hi
Sorry for this silly question.
Has anyone ever try to conjugate a biotinylated antibody with an Alexa fluor 488? What could be the limitations?
I know, this is strange but I would like to do a precise experiment that requires this combination. Basically I want to use the 488 as well as streptavidin 647.
Hi Loic!
I think that you can do it but with an streptavidin Alexa 488.
https://www.thermofisher.com/order/catalog/product/A32361
correct me anybody if I´am wrong!
@Oscar Recasens
I am already planning to use strepativin AF647 that will binds to the biotin. But in conjuction I would like to manually conjugate this antibody with AF488.
Basically, I have an biotinylated Ly6g antibody.
I would like to use the thermofisher conjugation kit to put AF488 on it. And then, in a second step, I will use this conjugated antibody and streptavidin AF647.
Your approach may be feasable, if the biotinylation level of your antibody is not excessive. In this case, free amino groups should be still available for a conjugation with a NHS-activated dye. Simply follow the protocol of the respective manufacturer of the fluorescent reagent. Please consider that the use of two dyes might lead to a FRET effect, which means that they do not emit independently.
Agreed with Michael G. Weller . If you need a dual detection of your antibody, you might use an anti-mouse Fc:AF488 or protein A:AF488 intead of performing the chemical conjugation?
I have a hidden reason for thinking of doing that way. I can't reveal it. However this reason is based on the use of a primary biotinylated antibody with a subsequent addition of streptavidin. But I would like also to have a fluorescent biotinylated antibody.
I know it can sounds silly but when you know why (things that I can't reveal yet) it makes sense.
Conjugate on the Fc-glycans! https://www.genovis.com/products/glyclick/
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