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

BATCH NUMBER 271685 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Double bands, MW is around 90.For human and rat samples, the bands from homogenate are Darker than from extract opposite for mouse samples. The bands from mouse samples are much darker than the others.

SAMPLE I tested the antibody with brain samples such as human homogenate, human extract, rat homogenate, rat extract, mouse homogenate and mouse extract.

PRIMARY ANTIBODY HIF1 beta ab2771, 271685. 1:1000 diluted, in 5% non-fat milk incubate for 18 hours,overnight, room temperature wash with 0.1% tween-TBS for 3 times, at 10 minutes each time

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED not used

ANTIBODY STORAGE CONDITIONS -20, but +4 degrees before use diluted (1:4) in 5%BSA.

SAMPLE PREPARATION in SDS buffer containing 2%SDS, 10%glycerol, 1%beta-mercaptoethanol and 0.0625M Tris-Cl

AMOUNT OF PROTEIN LOADED 10ug

ELECTROPHORESIS/GEL CONDITIONS 10% reducing

TRANSFER AND BLOCKING CONDITIONS buffer:1.4% glycine, 0.3% Trizma base, 20% methanol 1 hour block with 5% non-fat milk

SECONDARY ANTIBODY goat anti-mouse 1:5000 diluted in 5% non-fat milk for 2 hours at room temperature wash with 0.1% tween-TBS for 3 times, at 10 minutes each time

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? not altered

ADDITIONAL NOTES I checked literature and found HIF1 beta has several isoforms. Does ab2771 recognize only one isoforms or all?

I failed to attach the image of the film, which is jpg or doc format.

ANSWER:

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.

I have an inquiry about your product, ab2771; HIF1 beta antibody.

Which cell line did you use this antibody for Immunoprecipitation? Was it transfected cells?

The researcher wants to use this antibody for IP. And he found clone 2B10 except abcam wouldn't be recommended for IP.

Please advise us if you have any infromation about this purpose.

ANSWER:

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.

Antibody ab2771 came up after I queried the ABCAM database for antibodies against the T-complex 1 protein. Can you confirm that this antibody would react with this protein in human cells? Thanks

ANSWER:

Thank you for your enquiry.

ab2771 HIF1 beta/ARNT antibody [2B10] does not relate to the target "T-complex 1" protein. The search result using this phrase as a keyword does not retrieve anything specific.

Following a search of our on line catalogue I have located TCP1 alpha antibody [23c] (ab13501). This chaperonin protein shares many characteristics with the heat shock proteins. I encourage you to check out the datasheet by clicking on the link below to determine whether this antibody suits your requirements.

http://www.abcam.com/index.html?pageconfig=datasheet&intAbID=13501

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Customer Question: I need to know if this antibody is specific to any particular member of the ARNT family (ARNT1-3) or will it detect all family members.

ANSWER:

Thank you for your enquiry. This ARNT antibody detects the ARNT1 isoform, but it does not detect any of the other forms. Please contact us again if you require further assistance.