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dear sir, as per catalog no i have done my exp. but non availibility of substrate(missing) i have used p-nitrophenol phosphate as substrate & take reading at 540nm. reading shows: OD- 0.001, 0.02, 0.39, 0.04 . dilution factor 6. can you help me how to calculate the concentration of IL-beta. thanks & regards. --
Asked on Aug 15 2011
Thank you very much for contacting us. The standard we have provided in the KIT is 2 ng/ml; which is lyophilized. We recommend adding 2 ml of Diluent Concentrate (10X) in the IL1 beta standard vial which will give final conc. of IL1 beta 1ng/ml. We also recommend preparing the standard curve using serial dilutions of standard. The information is nicely explained in protocol book attached on the datasheet. The standard curve positive slope will show the OD of standard corresponding to the concentration of IL1 beta standard. Now place the OD reading of your test sample on the standard curve and try to calculate the IL1 beta concentration from graph. If you have diluted samples six times then multiply the final value of IL1-beta by 6. Now coming to the substrate used p-Nitrophenyl Phosphate substrate is commonly used with Alkaline phosphatase enzyme; in the Kit we have used Streptavidin-Peroxidase conjugate which I guess is a HRP enzyme. To give you idea of what exactly the peroxiodase enzyme is, I have also sent an email to lab for confirmation of the enzyme; I will send you the information soon. I am however sure that the enzyme is not a phosphatase enzyme. Could you specify why p-Nitrophenyl Phosphate was used? Finally the chromogen substrate should be there in the Kit however in case of missing components we always send these as free of charge replacements to our customers. Could you confirm if you have contacted Abcam scientific support team regarding the missing components and discussed matter with them? I hope my suggestion will help to improve your results. Should you have any other question please do not hesitate to contact me. I will also look forward to receiving your reply soon.
Answered on Aug 15 2011