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PC-12 nuclear extract lysate (hypoxia treated) (ab14886)

Submit a review Q&A (2)

製品の概要

  • 製品名

    PC-12 nuclear extract lysate (hypoxia treated)
    PC12 ライゼート 製品一覧

  • 特記事項

    This PC-12 extract (Hypoxia) was tested positively in Western blot and EMSA for NFKB p50 and Pax-3. Before harvesting the cells were deprived of oxygen for 4 hours. Extracts have been quality control tested by Western blot and the Electrophoretic Mobility Shift Assay (EMSA).

製品の特性

  • Mycoplasma free

    Yes

  • 製品の状態

    Liquid

  • 保存方法

    Shipped on dry ice. Upon delivery aliquot and store at -80ºC. Avoid freeze / thaw cycles.

  • バッファー

    pH: 7.90
    Constituents: 0.75% Potassium chloride, 0.009% PMSF, 0.01% Magnesium chloride, 0.008% DTT, 0.48% HEPES, 20% Glycerol
  • Concentration information loading...

  • 研究分野

  • 背景

    Generated by Greene and Tischler (1976) from a transplantable rat adrenal pheochromocytoma line, PC12 cells respond reversibly to nerve growth factor (NGF) and therefore have a neuronal phenotype, making the line a useful model system for neuronal differentiation. They produce catecholamines (dopamine, norepinephrine), neurotransmitters, NPP and are therefore used in studies of differentiation, neurobiochemistry and neurobiology.

プロトコール

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

参考文献 (0)

ab14886 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab14886 は論文での使用が確認できていません。

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Question

We stored the product according to the instructions for storing that came with the product. That is, aliquot it in small samples and froze them at -20, for the first 3 weeks and them transfer to -80. Either 4h or 16h of hypoxia should be enough to induce the expression of HIF in PC12, actually there are many papers in the literature that use only 4 h of hypoxia to get HIF induction. But the problem is that I could not see no even a very faint band even although we load 40 ug of your extracts. Moreover, as I told you in my previous message the nuclear extract of PC12 is the positive control recommended to test the antiHIF2a-ab199 antibody from ABCAM. Again I highly appreciate if you reconsider to dispense me of paying for a product that does not work as is meant to do.

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Answer

Thank you for getting back to us and providing further information. This PC-12 extract (Hypoxia) was tested positively in Western blot and EMSA for NFKB p50 and Pax-3. However, it has not been used to look for HIF-1a or HIF-2a. In terms of treatment, the cells were grown in chambers which were flushed with nitrogen gas for 60 seconds and then stored in carbon dioxide for 4 hours before harvesting. As for their hypoxic stability, they are stored in liquid nitrogen which should preserve their function. We can offer you either a credit note which you can use for your next order or a refund. Please do let us know how you would prefer to proceed. We look forward to hearing from you soon.

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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,

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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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