Anti-HIF1 beta antibody [2B10] (ab2771)

While we do have publications using this antibody in mouse samples we are not presently aware of specific publications using mouse brain homogenates. HIF1 beta forms heterodimers with HIF1alpha and has ubiquitous nuclear expression. The HIF1 complex rapidly degrades. It is critical to prep only a few plates/dishes/flasks of cells at a time and to immediately get the cells into ice cold buffers and perform the whole protein prep on ice. HIF-1 is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% or with treat using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical. Upon stabilization HIF-1 translocates to the nucleus. The best western blots (cleanest) are always done using nuclear extracts. It is possible to detect HIF-1 in whole cell extracts, but they tend to be much dirtier and the staining is much weaker. We recommend that a positive/negative control always be run side by side so that it is possible to discern which band is upregulated in the hypoxic sample. Depending on the sample, treatment, etc. you may see either a band or a doublet.

Thank you for your patience. I am sorry to hear that you are experiencing difficulties with this product ab2771 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. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. In most cases, the cause of multiple bands is because the primary and/or secondary antibody concentration is too high and causes non-specific binding. You can try decreasing the primary (1:1000 ~ 1:5000) and secondary concentration or run a no-primary control (without the primary antibody) to ensure that the multiple bands are not caused by your secondary antibody. Another possible reason is that the extra bands you are observing may be multimers. Please try boiling your protein in SDS-PAGE for 10 minutes to disrupt multimers. Meanwhile, bands located below the expected molecular weight you are observing may indicate that your target protein has been digested. Please make sure that you incorporate sufficient protease inhibitors in your sample buffer. The image you provided showed different strengths in expression. I would suggest doing optimization for each species sample separately, so that you can have better control of the level and intensity of the bands. This is because your target protein may exist in different amount in different species. I would also suggest incubating your primary antibody at 4 degrees instead of at room temperature. Incubation at room temperature is recommended to be done for 1-2 hours, while 4 degrees can be done overnight. Unfortunately, I do not have any data suggesting that this product can detect other HIF-1 beta isoforms at this time. However, I believe the following reference may help explain the double band that you are observing. Rescue of Hypoxia-inducible Factor-1 alpha-deficient Tumor Growth by Wild-Type Cells Is Independent of Vascular Endothelial Growth Factor. Can. Res. 62(10):2962-2970, 2002. http://cancerres.aacrjournals.org/cgi/reprint/62/10/2962 In this specific reference, the authors detect nonspecific cross-reactivity of the anti-ARNT polyclonal antibody in nuclear extracts of ES cells. Also, we see similar reactivity in the reference: Isoform specific expression of hypoxia-inducible factor-1alpha during the late stages of mouse spermiogenesis. Mol. Endocrinol., Vol 16: 234, Feb 2002. http://mend.endojournals.org/cgi/reprint/16/2/234 I hope the above suggestions and references will be helpful to you. If you still experience problems please do not hesitate to contact me with details of your order (purchase order number, shipping address/purchasing agent info, contact information, etc.) so that I can immediately arrange for a replacement or refund to you. Also, please advice on how you would to proceed with this enquiry. I look forward to hearing from you again in order to resolve the matter.

Thank you for your enquiry. Followingf corresponce with the source of this antibody I have determined that the cell lines used for IP were mouse Hep1c1c7 and human HeLa cells. ab2771 works well by IP in both mouse and human lysates at 1-2 ug/IP. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.