HRP Anti-Biotin抗体(ab6651)

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While it is theoretically possible to amplify your signal by using a biotinylated secondary antibody with an anti-biotin tertiary antibody, it is not commonly done. Because streptavidin binds to biotin with a very high affinity, it is very commonly used to provide amplification in biotin systems in IHC or ELISA. For example, you could use streptavidin-HRP (ab64269) to detect the biotinylated secondary antibody. When using biotinylated secondary antibodies in WB, it is important to block with BSA instead of milk. Milk contains endogenous biotins that can cause very high background.

To increase the WB signal, it is more common to use a more sensitive ECL reagent or a longer exposure. Alternatively, you could try things like enriching your samples by subcellular fractionation(ab109719), loading more protein, using more primary antibody, or incubating overnight in the primary antibody. Some researchers also produce a slightly amplified signal by using a tertiary antibody. For example, you could use an unconjugated rabbit anti-mouse (ab97042)followed by an HRP conjugated anti-rabbit (ab97051).

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for contacting us. I see no reason why anti-biotin antibody ab6651 or HRP conjugated-streptavedin protein ab7403 would not recognise the biotin conjugated to your customers protein of interest following a boiling step. Biotinylated protein ladders such as this one: http://www.cellsignal.com/pdf/7727.pdf are denatured by heating and then detected by anti-biotin antibodies. Either product should be applicable to your customers Western blot proceedure. No special proceedure should be required to perform the Western blotting. A general proceedure can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375 I hope this information has been helpful. If you have any further questions please do not hesitate to ask.