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I have got a question about second antibodies used for Western Blot. At the moment I am using Polyclonal Rabbit-anti-mouse immunoglobulins/HRP as second antibody. We would like to add a step for strengthening the signal, like using Biotin. Is it possible to use cat# ab97044 as second antibody in combination with cat# ab6651 for the detection with DAB? Thank you for your answer.
Asked on Jun 04 2012
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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.
Answered on Jun 04 2012