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I would like information about your agarose-immobilized anti-V5 antibody. The intended application is immunoprecipitation followed by western blot. My V5-fusion protein is ~55kb, so I am concerned about interference with the IgG heavy chain. Will the antibody remain bound to the agarose after I boil my samples in reducing SDS loading buffer (containing DTT)?
Asked on Dec 14 2005
Thank you for your enquiry. Following immunoprecipitation, some heavy and light IgG chains may be eluted with reducing buffers such as Laemmli. This may cause interference with your V5-tagged protein. The best and cleanest approach would be to IP using a V5 antibody raised in one species (e.g. mouse) and perform a western blot using an antibody raised in another (e.g. rabbit). I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
回复于 Dec 15 2005