Anti-HIF1 alpha antibody [H1alpha67-sup] (ab463)

Dear technical support team:

This customer has highly interested in ab463, ab55878 and ab117115, she would likes to order these antibodies and test in IHC-P with dog sample, would you please help this customer to createthree discount code.

Thanks for your kindly help.

ANSWER:

Thank you for your reply.

Thank you for agreeing to test these products in IHC-P in dog samples. We look forward to receiving your Abreviews.

Please don't hesitate to contact me should you have further questions.

Product code: 463

Inquiry: Dear technical support team: This customer raises an enquiry about ab463 (Anti-HIF1 alpha antibody [H1alpha67-sup]) and ab55878 (Anti-Beclin 1 antibody), this customer wants to conduct IHC-P with dog sample, and therefore she wants to know what percentage the sequence identity is between the immunogen of these antibodies and dog’s sequence. If the sequence similarity are too low, could you please offer any suitable selection which can conducted IHC-P with dog sample to her? Thanks for your kindly help. Best regards

ANSWER:

Thank you for your enquiry.

I am sorry to confirm that as far as we are aware, ab463 (Anti-HIF1 alpha antibody [H1alpha67-sup]) and ab55878 (Anti-Beclin 1 antibody) have never been tested with samples from dog. All tested species cross-reactivity information is stated on our datasheets, and these are updated as soon as any new information is brought to our attention.

ab463:Theimmunogen information is on the datasheet: amino acids 432-528 of Human HIF 1 alpha. This corresponds to the followingsequence:
DVMLPSPN EKLQNINLAM SPLPTAETPK PLRSSADPAL NQEVALKLEP
NPESLELSFT MPQIQDQTPS PSDGSTRQSS PEPNSPSEYC FYVDSDMV

I have conducted an alignmentand canconfirmthatthisimmunogen sequencehas 90% alignment with the dog sequence. It is therefore likelythat this antibody woulddetect in dog samples. However, I am sorry we are not able to provide a guarantee for the results from dog until it has been tested.

ab55878: The immunogen information is provided on the datasheet:
RLVPYGNHSYLESLTDK
I am sorry I am regrettably not able to obtain the dog Beclin 1 sequence from the protein database sitesto check the alignment in this case. I would be pleased to run the alignment for the customer if they are able to provide the dog sequence?

If the customer would like to test ab463 HIF1 alpha antibody in dog samples, please contact me again by replying to this message prior to the purchase as you may be eligible for our testing discount program.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

http://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact me for any further advice or information in this regard.

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.

Thanks for your kindly reply, after contacting with this customer and passed these suggestions to herm then she modified the experiment step as your suggestions, but it did not improve the result. I also attached the result in this letter, please check it and help this customer to solve these problems.
Thanks for your kindly help
Best regards

ANSWER:

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

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

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Dear technical support team, Our customer raises an enquiry about ab463 (Anti-HIF1 alpha antibody [H1alpha67-sup]), according to the online datasheet this antibody's positive control can be used Glioblastoma multiforme this customer wants to know is there any suggestion can help she to choice another positive control? Could you please help this customer to solve the problem? Thanks for your kindly assistance. Best regards

ANSWER:

Thank you for your email.

There are indeed several positive controls which can be used. It is noteworthy, that this protein is induced when cultured in hypoxia (with less oxygen). Please see additionnal protein information also on SwissProt http://www.uniprot.org/uniprot/Q16665.

In tissue, the levels are highest in heart and kidney. If the customer is using cell lines, please find here a list of cell lines in which HIF1alpha has been detected. Note, induction through hypoxia increases the protein levels and I can therefore recommend this.

Cell lines in which HIF1 alpha has been observed:
-DFO-treated HeLa cells
-hypoxically or CoCl2 induced cell line such as MCF-7,PC12, 293, or MEF
-CoCl2 induced in Ramos cells
-CHO, HeLa and K562cells

I hope this information is helpful. Please do not hesitate to contact us again, should you have any further question.