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Thank you for your e-mail. I have some questions about your Ab412-200. I purchased your Ab412-200. The lot No. 212546. I am planing to perform immunoprecipitation by using it. I want to know the concentration of this antibody. To measure the concentration of this antibody, I diluted normal mouse IgG and this Ab412-200 and then performed western blotting to compare the concentrations of these two antibodies. I detected two bands of Ab412-200 which seemed to correspond to the heavy chain of normal IgG and two bands of Ab412-200 which seemed to correspond to the light chain of normal IgG. I am confused. I want to know the concentration of the Ab412-200 and how much Ab412-200 I should use for immunoprecipitation. I have HEK293 cells. Because I want to set up negative control for immunoprecipitation by using the same amount of normal mouse IgG. I am looking forward to your reply.
Asked on Aug 17 2006
Thank you for your enquiry. This antibody is an ascites preparation. Ascites contains proteins aside from the antibody of interest. Ascites fluid is intraperitoneal fluid extracted from mice that have had hybridoma cells expressing specific monoclonal antibodies injected into their peritoneal cavity, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and secrete high-titered antibodies. Ascites fluid is clarified by centrifugation to remove the lipid layer and the cell pellet. It contains specific antibody as well as other host serum proteins including immunoglobulins. While the total protein concentration is ~20 mg/ml, the specific antibody concentration range is 5-10 mg/ml. An exact antibody concentration is only relevant for purified antibodies. For IP we recommend using 10-500 µg cell lysate plus 0.5-5 µg of antibody. Therefore whilst I cannot give you a specific dilution for IP as this should ultimately be determined by the end user I would recommend that you apply the antibody at a dilution of 1/1000 if you are using a 1ml lysate/buffer mix; thereby presenting a good starting mass of ~5-10ug. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Aug 18 2006