Question (11178) | PC12 nuclear extract lysate (hypoxia treated) (ab14886)

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Question

I purchased very recently from Abcam the product PC-12 hypoxia nuclear lysate- ab14886. We ordered the product at the end of November and received it at the beginning of January. Last week we used this proteins for the first time loading 40 micrograms of them in a SDS-gel to analyze them by western blot with an antibody for HIF2alfa (Abcam- ab199) and no band was seen with this antibody. I purchased those proteins (ab14886) just to use them as control in some experiments that I was doing in PC12. But, by the time that the product came to the lab I had worked out the conditions of my proteins extraction protocols such that my PC12 proteins (obtained from cells maintained in normoxia and to a higher extent after hypoxia) now showed a clear and very strong band (16h of hypoxia) when inmunoblot them with the HIF2alpha antibody (ab 199). We did run in parallel, the PC12 proteins extracted by my technician in my lab, and those that I got from Abcam, and after immunobloting with antiHIF2alpha only a band was seen in the homogenate of proteins extracted in my lab, indicating that there must be something wrong in the PC-12 hypoxia nuclear lysate (ab14886) that we received from Abcam. I am really surprised of the lack of results with this product since it was specifically indicated as the positive control in the datasheet that came with the ab199 antibody. For all this, I have given instructions to stop the payment of this product until I get a convincing explanation or a new product. I´ll be waiting for your decision,

Answer

Thank you for your getting back to us and for providing further information about your experiment. Firstly, we would like to know how this lysate was stored at-80°C in your lab? We would like to emphasize that there is a difference in the way cells were induced in your lab (16 hours oxygen deprivation) and our cells (4 hours oxygen deprivation) which could lead to some differences in the expression of some factors (please look at the datasheet). Moreover, so far we have not tested yet this nuclear extract for Hif-2. We hope this will be useful for you. If you need anything further or any help then please let us know.

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