Anti-Aurora B (phospho T232) antibody (ab61074)

Having difficulties with background wtih ab61074
Mosue samples
What protocol was used to test in WB?

ANSWER:

Thank you for taking the time to telephone us with your enquiry yesterday afternoon. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

As discussed on the telephone, I have contacted our source for this antibody to obtain the testing protocol for you. Also as discussed, unfortunately this antibody has not been tested an guaranteed for use in mouse samples. I can therefore recommend to include a human positive control sample in the experiments, which would be covered by the guarantee.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.


Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


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


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Thank you for your email.


Our customer would like to receive a credit note.

Please arrangethis.





Best regards

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: xx

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

Testing discount code to try antibody in IF.

ANSWER:

DISCOUNT CODE: *******
Expiration date: *************

I am very pleased to hear you would like to accept our offer and test ab61074 in IF in human tissue. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IF in human tissue and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Our customer explained his data showed Nocodazole treatedHela cells and P-H3 used as a positive control.
Because he set P-H3 as a positive control and checked bands on 8˜9 hr, he thinks p-Aurora B should detected.
Please let me know if you need more information.

Best regards,

ANSWER:

Thank you for liaising with the customer and updating us. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed the protocol details with the confirmation of the cell lines used, 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 of the same batch (we do not have a new batch in stock), an alternative antibodyor a credit note.

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

DESCRIPTION OF THE PROBLEM No signal or weak signal
SAMPLE HeLa cell (human)
PRIMARY ANTIBODY Concentration or dilution 1:1000 Diluent buffer 3% BSA TBST buffer Incubation time over night Incubation temperature: 4℃ Buffer TBST Number of washes 3times
DETECTION METHOD ECL, Chemi doc
POSITIVE AND NEGATIVE CONTROLS USED -
ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION Lysis buffer RIPA buffer Protease inhibitors: protease inhibitor mixture Phosphatase inhibitors : NaF, NaVO4 Reducing agent : DTT Boiling for ≥5 min? yes
AMOUNT OF PROTEIN LOADED 60ug
ELECTROPHORESIS/GEL CONDITIONS gradient gel (bio-rad) 10% gel
TRANSFER AND BLOCKING CONDITIONS Type of membrane NC 0.25um Protein transfer verified ponceau S Blocking agent and concentration 5% BSA Blocking time 30min. Blocking temperature RT
SECONDARY ANTIBODY Secondary antibody: Bethyl, Cat no A120-501P Species: Goat Reacts against: rabbit Concentration or dilution 1:3000 Diluent buffer 3% BSA TBST buffer Incubation time 2hr Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP conjuagated Buffer TBST Number of washes 3times
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? -
ADDITIONAL NOTES -

ANSWER:

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

Having reviewed this case, I would like to offerthe following suggestion: I would also appreciate if you can confirm some further details:

Indeed, the antibody is against the phosphorylated Aurora 2 in T232. Often this modification needs to be induced. Does the customer know that this phosphorylation occured in the Hela cells he used? Have the Hela cells been treated? Else I can stronly recommend to use a positive control. As positive control, I can recommend for example extracts from COS7 cells treated with Nocodazole (see our datasheet), orOE21, Kyse-410 and OE33 cells accordingly to the reference http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3094318/?tool=pubmed

Also in the below linked publication, the autors show that in Hela cells, the Aurora T232 can be induced with for example transfection of Sds22. http://jcb.rupress.org/content/191/1/61.full

Please let me know the exact conditions of cells the customer used. As said, I can recommend to use cells with an high level (induced) pof Aurora T232.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet, and the product isstillunder guarantee,we would be pleased to provide a free of charge replacement or a credit note.

I hope this information is helpful, and I thank you for your cooperation.