Anti-NALP2抗体(ab36850)

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab36850 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 credit note/refund will be offered. After looking at the protocols you used, I am surprised that even 100ug 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. - what type of cells are you are using? Are they known to have sufficient levels for detection? The positive controls we recommend is PC-3 cell lysate. It would be useful to perform a simple WB with the positive controls to see if you get a band at the correct molecular weight. The weak signal that you managed to obtain, was it at the 96-121kDa band area? - is the lysis buffer you used as strong as RIPA buffer? 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. - what blocking agent did you used and for how long? We recommend using 5% BSA for 1 hour. The reason why you are not seeing any bands might be because cross reaction between the blocking agent and antibodies might have occurred. Please try 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? Was the secondary antibody HRP conjugated? What is iBlock? Is it a commercial diluent? Has its performance been shown to work well with other experiments? Cross reaction between the antibodies with this 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. - was the incubation of the primary antibody done overnight at 4oC? Incubation at this temperature and for longer duration will ensure proper and sufficient binding to the protein of interest. - can you confirm the washing buffer concentration? How much Tween was in the PBST solution? In general, a 0.05% PBST would be sufficient as a washing buffer. This means in a 1000ml PBS, you will add 500uL of Tween. Also, was the washing done 15min x 3 times? This is because over washing will eventually cause you to lose signals. - lastly, can you confirm the detection method used? At Abcam, we recommend using an ECL+ or super signal kits from pierce as they are much more sensitive. A poor quality detection solution will result in low signals. I hope the above recommendations may already help you, if you still experience problems please do not hesitate to contact me with the answers to the above questions and an image of the weak signal. I would also appreciate if you could also provide details of your order (batch number, purchase order number, shipping address, contact information, etc.) so that I can immediately arrange for a replacement or refund for you.