HIF1 alpha antibody panel (ab10625)

Dear All,

I have a special request for you I hope to be more clear as possible to better explain you this issue.

Our customer has ordered ab463 last year form Euroclone. But this antibody did non work in WB analysis.

Please note that the same antibody was already purchased by a colleague of our customer in 2005 and it did not work (please see attachment where the customer wrote to your customer service), so you have replaced it.

As you can see in your datasheet (in attachment), it was never updated as WB application still remains!!

The customer told me that you have replaced ab463 purchased from other italian customer, so she is asking if you can send a free vial of ab1 (as you have suggested).
Anyway our customer has also filled the form for the complaint (see 3rd attachment)
Please let me know if you can send us a free of charge vial of ab1.

Thanks for your support
Kind regards

Antibody code: HIF alpha antibody panel (ab10625): Mab1-ab463 HIF alpha mouse monoclonal antibody; clone HIF alpha67; Isotype=IgG2b; Purity=Protein G. Concentration=2,0 mg/ml
Lot number: GR34677-1
1) Antibody storage conditions (temperature/reconstitution etc): + 4 °C

2) Description of the problem (high background, wrong band size, more bands, no band etc.):
wrong band size (60 kDa).

3) Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.):
nuclear extracts from human oral squamous cancer cells.

4) Sample preparation (Buffer/Protease inhibitors/Heating sample etc.): as reported in the datasheet
in the section 'Protocol for HIF alpha antibody panel'. Before loading, samples were boiled at 100&oC for 10 min.

5) Amount of protein loaded: 40 mg

6) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.):
SDS PAGE in denaturing conditions, gel 12%, run: 100V.

7)Transfer and blocking conditions (Buffer/time period, Blocking agent etc.): Transfer to
nitrocellulose membrane was performed in Transfer Buffer (25mM Tris-HCl, 200 mM Glycine, 20% MetOH) for 1 h , 100 V constant. After the transfer, a visualization of proteins was performed using Ponceau Red. Blocking was performed incubating the blot for 2hs in 1X TPBS (1X PBS, 0,1% Tween) 5% milk under gentle agitation.

8) Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Mab1-ab463 HIF alpha antibody was incubated 1:2000 overnight at 4°C, under gentle agitation; after incubation three washes of 10 min in 1X TPBS were performed under gentle agitation.

9) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Secondary anti-mouse IgG HRP-linked antibody from Cell Signalling was incubated 1:2500 for 1 hour at RT; 3 washes of 10 min in 1X TPBS were performed under gentle agitation.

10) Detection method (ECL, ECLPlus etc.):
The Immuno-Star WesternC Kit form Biorad was used as ECLPlus for the detection.

11) Positive and negative controls used (please specify):
One sample treated with the hypoxia mimetic Deferoxamine was used as a positive control

12) How many times have you tried the Western?:five times

13) Have you run a No Primary control?:no

14) Do you obtain the same results every time?: yes
e.g. are the background bands always in the same place?: yes

15)What steps have you altered?:
Primary antibody was incubated at a lower dilution (1:500)

Additional Notes:
As reported in the answer to the Question 15 in the Section Scientific supports in the datasheet of the Anti-HIF1 alpha antibody [H1alpha67-sup] (ab463), (“We recently updated the online datasheet for ab463 to state that the antibody is unsuitable for Western blotting due to some customer complaints that we received. Instead of ab463, we recommend using ab1 for Western blotting. Ab1 is: Mouse monoclonal [H1alpha67] to HIF1 alpha”) this antibody is unsuitable for Western blotting but the datasheet has not been updated.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from the ab463 Anti-HIF1 alpha antibodyfrom this panel.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee forWB and human samples.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results.

I apologise for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

With regards to the WB application on the datasheet for ab463, according to our records the WB application was originally removed as we were concerned about how well it works in WB. However, we have since received more positive feedback on the use of ab462 in WB, including an Abreview from a customer published on the datasheet, and so WB was added again to the datasheet in January 2009. Also, HIF1 alpha is known to be a difficult target fordetection by any antibody, and it is not unusual for experiments to require a lot of optimization.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of this antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, we would be pleased to provide a free of charge vial ofab463 or ab1, which will still be covered by our guarantee. Please note that these are both the same antibody clone, and as previously discussed this is a difficult target for detection. Alternatively, I fully understand your concerns and you prefer a credit note in this case, I will be pleased to arrange this for you.

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

I need an anti-HIF antibody that will work in IHC-Fr on mouse tissue. I also need positive controls for this.

ANSWER:

Thank you for contacting Abcam.

I have had a chance to look through the data for at HIF1 alpha antibody panel (ab10625) that you had spoke of during our conversation. Of the antibodies included in this panel only ab8366 (Anti-HIF1 alpha antibody [ESEE122]) has been tested to work in IHC-Fr. Although the other antibodies may work in with this condition they have not been tested as such and therefore I cannot be confident that this product will meet your needs completely.

As such I would recommend using ab8366 for your experiments. Positive controls for this product include glioblastoma multiformae and hypoxia-induced human placenta.

I would also like to highly recommend ab1 (Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade). This product has been tested in IHC-IF on mouse and has been referenced in 33 publications. The Abreviews for this product may also be an excellent resource for your experiments. I've included links to both these antibodies as well as the direct link to the IHC-Fr Abreviews for Ab1 below.

ab8366: http://www.abcam.com/hif1-alpha-antibody-esee122-ab8366.html

ab1: http://www.abcam.com/HIF1-alpha-antibody-H1alpha67-ChIP-Grade-ab1.html

ab1 reviews: http://www.abcam.com/index.html?datasheet=1&tab=abreviews&intabreviewid=9511

I hope this information helps you. I would also like you to know that these products are covered by our Abpromise®. Which ensures that you can trust our products to perform as stated on the datasheets, or we will offer a replacement, credit, or refund.

The Abcam Abpromise® guarantee: • 100% Scientific and Customer Support for any product you buy from Abcam or one of our authorized distributors. • We guarantee our products work in the tested species and applications stated on the datasheet. • We will replace or refund products not performing as stated on the datasheet if reported within 6 months of purchase. • We investigate any quality concerns raised by customers to ensure our catalog contains products that perform to the highest standards.

Please note these conditions to our Abpromise®: • Protocol information must be provided in order for the claim to be validated. • Antibodies tested in recombinant samples only are not guaranteed for use on endogenous samples. • “Predicted to react” information on the datasheets is provided for reference only and these species are not guaranteed. • Fast Track antibodies are covered for ELISA against the immunizing peptide only. I hope this information is helpful, but please do not hesitate to contact us with further questions.

thanks for the detailed reply and sorry for the delayed response to your email.I would be most interested in arranging some form of try before you buy scheme regarding the antibodies for Hif 1a, and would be more than willing to give feedback concerning cross reactivity in return for a discount.I think it would be most beneficial to all concerned if we could arrange for this to incorporate an antibody panel for hif1a e.g. ab6679 and ab10625. I suggest this as more conclusive information can be obtained for both parties concerned. Since at present no current information exists for reactivity with invertebrate samples, thus there is no defined or logical starting place to begin an antibody test, other than a simple broad scale testing program.I would be in the position to give feedback regarding reactivity within a species (embryo & adult), and between species also.Please reply by return and let me know if you would consider this as a feasible arrangement.

Thanks in advance Steve

ANSWER:

Thank you for your enquiry.

We have found that certain HIF antibodies work in one person's hands but not in another, and vice-versa. The ab10625 contains 2 different HIF abs that work in IHC and the ab6679 contains 4 different HIF abs that work in WB. Both of these kits were designed for the purpose of providing the customer/researcher with different samples to try.

Therefore, we are very sorry to inform you that we do not provide samples of either of these kits and they have already been offered at a discounted price.

BATCH NUMBER 131C/123 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No band on Western Blotting at 120kD in human endothelial cells treated with CoCl2. The identical samples give a strong positive band at 120kD using the BD Pharmigen antibody. Several non-specific bands are seen with lower molecular masses. I had wanted these antibodies to detect HIF-1alpha in mouse cells, but no band was visible in hypoxia-treated mouse cells either (cannot compare this with the BD antibody, as this does not react with the mouse protein). Also no reactivity with hypoxia-treated mouse or human cells on immunofluorescence. BD antibody does give some positive IF with human cells.

SAMPLE Human endothelial cells treated with CoCl2, whole cell extract. Mouse neutrophils, hypoxia-treated, nuclear extracts, Whole human neutrophils Hypoxia-treated, cytospun and fixed with paraformaldehyde and cold methanol for immunofluorescence. Mouse neutrophils similarly treated for IF.

PRIMARY ANTIBODY primary antibody (1:250) overnight at 4oC, washed 4x with PBS/tween,

SECONDARY ANTIBODY then secondary antibody (DAKO sheep anti mouse IgG, 1/5000) for 2h at room temp.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Positive control was a verified sample of HUVECs treated with CoCl2, that gives a very strong band at 120 kD with BD Pharmigen antibody.

SAMPLE PREPARATION Western blotting: Endothelial cells washed and lysed in Reducing Sample buffer with SDS and Mercaptoethanol, plus protease inhibitor cocktail. Heated for 5 min at 95oC and run immediately or stored at -20oC.

TRANSFER AND BLOCKING CONDITIONS Blotted onto PVDF, blocked for 2h with 10% milk,

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

WHAT STEPS HAVE YOU ALTERED? Have played extensively with the extraction methods, altering lysis buffers, changing sample heating times. Have also tried the ab1 antibody (NB 100-105), and that did not work either.

ADDITIONAL NOTES Background bands appear to always be in the same place regardless of the experiment, although they differ with the two cell types (endothelial cells or neutrophils).

ANSWER:

I'm sorry you are having problems with this kit and ab1, and take your complaint very seriously. I would like to know if those products were shipped to you together, and whether they were bought from a distributor or through Abcam directly. This information will greatly help me,

I look forward to hearing from you,