Anti-DDX3抗体(ab37160)

Thank you for your enquiry. I am sorry to hear you are having a problem with ab37160. I would like to suggest the following modifications to your protocol: -Run a positive control of HepG2 (Human hepatocellular liver carcinoma cell line) whole cell lysate (we have some in our catalogue if you need to purchase this, reference ab7900) as it may be that your samples express small amount of the DDX3 protein, which are not easily detected in western blotting. -As you have not specified the type of lysis buffer you use, I would like to make sure you use a strong buffer, such as RIPA buffer, to extract the protein, as it is located predominantly in the nucleus. It may be that a nuclear extraction procedure is needed to concentrate the protein in your samples. I would be happy to provide you details of those protocols if you need those, please do not hesitate to ask for them. -Incubate the membrane with primary antibody overnight, at 4C, to maximise the binding of the primary to the protein. -Use an ecl+ or superSignal detection kit, as these are more sensitive than Ecl and will give you stronger signal. -The antibody has only been tested in reducing conditions, therefore it may be that it only works in those conditions. I would recommend switching to a reducing gel and also making sure the conditions are denaturing too. -I am not sure if you mean you block overnight, or transfer overnight. I would recommend to block only 1 hour at room temperature, and trying both 5% milk and 5% BSA as some antibodies prefer one blocking agent over another. -Please also make sure the secondary antibody works well with other primary antibodies, as it may be loosing activity and not be working well. Please let me know if this helps and do not hesitate to contact me again if you still experience problems with the positive control and the recommended changes. I would be happy to offer you a replacement vial or refund if the antibody was bought in the last 120 days and it still does not perform after those tips.