Hello. TetraHis antibodies recognise 4xHis-tag in the protein. What if I have a long His-tag (e.g. 10xHis-tag) and I perform ELISA or Western Blot based on native PAGE? Is there a chance that two TetraHis antibodies will sometimes bind to the long His tag and I'll get a false signal increase? Or is the potential distance between the antibodies too small and therefore after the first molecule has bound, the second molecule won't be able to approach the His-Tag, and it will be always 1 binding per 1 protein?
I started to think about it because the manufacturers of precoated well plates indicate the amount of histidines in the tag of the target protein. Is it possible that they imply potential overbinding?
Thanks.
Hello, on my oppinion, if you are only working with that "long"Histag protein, is not relevant how manny antibodies are allowed to bind to the tag, because they are to bind the same amount (mean) to each singular protein.
You have to realized that your are not working with 1 single protein molecule, you have thousands units of your protein, so what you are measuring is the average of thousands signals (including the one with pottentialy 2 bind Ab and the one with 1 binded Ab). So yes, i think that if you are measuring a signal increase, it is a real increase in what you are measuring.
I hope this help you
Greetings
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