Anti-Bak 抗体 [Y164] (ab32371)

The concentration of 1542-1 lot YI063010CS is 0.1160 mg/ml.

Thank you for contacting us.  The purpose of the blocking step is to prevent non-specific binding between the antibodies and the tissue section, membrane, 96-well plate, etc.  It is possible to get a good signal without a blocking step, but blocking will help increase confidence that your staining is specific and will improve the quality of your images.  If you observe signal when doing an isotype control, that would indicate that you are detecting non-specific signal that could be prevented by blocking.  To block tissue sections we recommend incubating them with blocking solution just prior to applying the primary antibody.  The blocking should be done after any antigen retrieval or permeabilization.  Using 10% normal serum from the host species of your secondary antibody will allow any regions of the tissue that would bind to the primary or secondary antibodies to be coated with antibodies that will not generate signal.  For example, if you are using a goat anti-mouse secondary antibody, we recommend blocking with 10% normal goat serum.  Additionally, depending on what type of detection you are using, further blocking steps may be necessary.  When using an HRP conjugated secondary, it is recommended to use an H2O2 pre-treatment to quench endogenous peroxidases.  Similar pretreatments exist for AP and Biotin.  There are also additional blocking steps that can be done when autofluorescence is a problem.  For more information about additional blocking steps, please see the following reference: http://www.ihcworld.com/_intro/ihc-methods.htm I hope this helps, please let me know if you have any further questions.   

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab32371 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. After looking at the protocols you used, I am surprised that even 50ug of protein, you still did not manage to get any bands. However, it is very hard for me to determine the cause without more details of your WB protocol. I would therefore appreciate if you can please clarify the following items. - Was the Triton X lysis buffer you used a RIPA buffer? If not, please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out. - could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. - Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. - can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. - Cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Can you confirm you have reduced and denatured the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol? This will ensure the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order (purchase order number, date of purchase, shipping address/purchasing agent, etc.). Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

Thank you for your enquiry. The laboratory that tested this antibody in IHC has provided the detailed protocol and I have added this to the datasheet of the antibody for your convenience. You can access this via the datasheet link below. Please do not hesitate to contact me if I can be of further assistance,