Anti-p53 (phospho S15) antibody (ab1431)

Catalog number(s): 1431

Product(s): Rabbit polyclonal to p53 (phospho S15)

Survey results
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Telephone section
Q. Did you contact Abcam's Scientific Support by telephone in reference to this complaint?
A. Yes
Q. Was it clear from the phone options which number you needed to press to get your issue resolved?
A. Yes

Protocol advice section
Q. Did Abcam's Scientific Support give you protocol advice?
A.Yes
Q. How did you feel about being given protocol advice?
A.I requested protocol advice
Q. Did the product work successfully after following the advice?
A.No
Q. Why have you not tried the protocol advice? (Select all that apply.)
A.

Free of charge replacement section
Q. Did Abcam's Scientific Support give you a free of charge replacement?
A. Yes

Q. Did the replacement product work successfully?
A.No
Credit note / refund section
Q. Did Abcam's Scientific Support give you a credit note or a refund?
A.Yes

Q. Would you have preferred a different outcome from receiving a credit note or refund?
A. Availability of a second Ab choice for mouse tissue
Overall satisfaction section
Q. How satisfied are you with how the complaint was handled?
A.Very satisfied
Q. Could we have done anything to better resolve the problem?
A.The tech was great with helping me trouble shoot possible issues.
Q. Would you mind if our scientific support team contacted you to better understand what happened and reach an agreeable solution?
A.No, I would not mind
Q. Please share with us any final thoughts you may have about Abcam, the complaints process, or our products.
A.

ANSWER:

Thank you for your participation in Abcam's post complaint survey.

I am glad to hear that your satisfied with our service. Although the outcome of a working antibody is always prefered,unfortunately, we needed to refund you purchase as the free of charge replacement did not work either.

You mentioned in your response "Availability of a second Ab choice for mouse tissue" as a prefered outcome. Could you please briefly explain what you specifically refering to. We do have a large selection of secondary antibodies, but maybe not what you needed. Please let me know and I can look into this further.

If there is anything else I can do for you, or if you have questions to any of our products (e.g. primary antibodies, lysates, secondary antibodies, kits, controls, gels, proteins/peptides, slides, biochemicals, conjugation kits), please let me know.

At the moment I am trying to investigate the p53 activity in my cell line using the western blot technique. I am using a antibody for total p53 and a phospho-p53 (Ser15) antibody. I ordered those antibodies from a different company. However I am not completely happy with those antibodies. Therefore I am looking into ordering new antibodies.

I was wondering which are the conditions you optimised your p53 antibody (ab26 or ab28) and phospho-p53 (Ser15) antibody (amount of protein needed, Blotiing solution and time, etc.)? How long would the shipping be taking?

ANSWER:

Thank you for contacting us.

Our p53 antibody ab26 has been optimized by loading 20 ug per lane of HeLa cells, using 5% BSA as the blocking agent for 1 hour at room temperature, and incubating with the primary antibody at a concentration of 5ug / mL overnight at 4C. To view the protocols used by our customers, you can browse our WB Abreviews for this product:

http://www.abcam.com/p53-antibody-PAb-240-ab26-reviews.html#WB

Ab28 and ab1431 can be used with a similar protocol, loading 20-40 ug mer lane, using a 5% milk block, and a primary antibody dilution of 1:1000.

We will ship anywhere in the UK or US overnight for next day delivery. I hope this helps, please let me know if you need any additional information or assistance.

IHC-P with mouse testis
previous lot in 2010 worked great
recent lot did not, received free of charge replacement
replacement lot (GR38575-5) not working either: no staining
Ab: 1/5, 1/10, 1/20
block: goat serum
AR: citrate, EDTA
secondary: Alexa, works well
checked previous tissue with new lot - not working

ANSWER:

Thank you for contacting us.

Your credit note ID is xxx.

I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

I would like a Certificate of analysis for:  ab1431

ANSWER:

Thank you for contacting Abcam.

Attached please find the certificate of compliance you requested.

I hope this information is helpful.  Please do not hesitate to contact us if you have any additional questions.

LOT NUMBER 691860 ORDER NUMBER 511808

DESCRIPTION OF THE PROBLEM No staining

SAMPLE Mouse lung tissue with tumors

PRIMARY ANTIBODY I tried 1:15, 1:25, 1:50, 1:100, 1:200 for one hour followed by a wash buffer rinse

DETECTION METHOD DAB+ (DAKO) for 5 minutes

POSITIVE AND NEGATIVE CONTROLS USED Should have internal control

ANTIBODY STORAGE CONDITIONS 4 degrees Celsius

FIXATION OF SAMPLE 10% NBF overnight

ANTIGEN RETRIEVAL 25 minutes in a steamer with hi pH - 9.0 (from Dako)

PERMEABILIZATION STEP N/A

BLOCKING CONDITIONS Serum-free protein block (Dako) for 5 minutes

SECONDARY ANTIBODY I used an anti-rabbit labelled-polymer (Dako) for 30 minutes followed by two wash buffer rinses

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I kept knocking the dilution down.

ADDITIONAL NOTES There is absolutlely no brown staining - not even a hint of possible background or non-specific staining. I ran each dilution once. I started with 1:50, 1:100, 1:200 on one day I tried 1:25 the next day, and 1:15 the next day. I ran them on the Dako Autostainer along with other slides that would have required the same secondary, and they stained fine.

ANSWER:

Many thanks for sending to us your full protocols for IHC with ab76242 and ab1431, these were very useful and I was able to examine your problem rapidly.

I think in general your protocol looks very good and I identified 2 possible reasons for the lack of signal, when I compared your protocol with that of the laboratories which tested the antibodies.

Firstly, the laboratories have not tested the antibodies with an antigen retrieval method of pH9 buffer, but have used citrate buffer pH6. This difference in buffers may explain that the epitopes are still masked with your method, and hence are not being detected by both antibodies.

Secondly, I noticed that the protein target phospho p53 is found in many locations (cytoplasmic, endoplasmic reticulum and nuclear) and as you do not have any permeabilization agent with the wash or antibody dilution buffers, the antibodies may be currently struggling to enter these compartments.

I would therefore recommend to change the antigen retrieval buffer to pH6 and to add 0.1% Tween-20 or 0.3$ tritonx100 in your wash and/or dilution buffers.

If you are still experiencing problems following those changes (you may have to try different dilutions of antibody to optimise the concentration) please do not hesitate to call us or e-mail us back and we will be happy to offer you a replacement or refund on those products.

Best of luck with your next experiments.