Anti-DcR3抗体(ab8405)

Thank you for your e-mail and your patience. I have reviewed your protocol as well as the correspondence you have had with my colleague Tom and I understand that you have had problems with ab8405 after an immunoprecipitation step or with a simple western blot. It is strange that with an IP you pick up a band in reprobing but with a standard western you see no bands. Unfortunately we are not sure we understand the procedure you are following in the IP experiment and would very much appreciate if you could clarify what antibody you are using to IP DcR3. The antibody has not been tested itself in IP so we are wondering if this may be the problem. If you have used the antibody ab8405 to reprobe using another antibody I would like to suggest you contact the supplier of the antibody as the problem may lie with this product. If you have used the antibody previously and it is the recent lot not working could you please provide the lot numbers so I can see if they come from the same rabbit? The antibody should work well in western blotting and I would be happy to offer you a replacement vial or refund if the antibody does not work well in this application if the problem is not protocol related. It is very hard for me to determine this without more details of your WB protocol. I would therefore appreciate if you can please clarify what type of cells you are using as I wonder if the samples have sufficient levels of DcR3. The positive controls we recommend are human brain, liver or kidney. It would be useful to perform a simple WB with one of those positive controls to see if you get a band at the correct molecular weight with ab8405. If your samples are know to have high levels of DcR3 or you have a good positive control the problem may be due to the antibody or a step in your protocol and I'd be very grateful if you could answer those few questions to help me understand the problem better: -what do you mean by standard preparation of samples: can you please confirm the lysis buffer you use, that you boil samples in reducing loading buffer as this may be the root of the problem -what dilutions have you tried? What incubation time have you tried with ab8405? I would recommend trying 1/500 and overnight incubation if you have not done so already. -what blocking agent have you used (and percentage) and for how long? Did you add it with the primary antibody? This can affect greatly the affinity of the antibody to its target protein in WB and we recommend using 5% BSA for 1 hour and incubating the antibody in TBST only. -we sometimes see migration problems with pre-made gels which are old; could it be that the gel you used is close to its expiration date? -does you secondary antibody work with other primary antibodies? We recommend using an ecl+ or super signal kits from pierce as they are much more sensitive or super signal kits from pierce as they are much more sensitive. I hope the above recommendations may already help you, if you still experience problems with those please do not hesitate to contact me with the answers to my questions and I will be happy to help you resolve this matter.