Anti-p53 antibody (ab2433)

Thank you for the advice with different experimental conditions. We already spent a number of hours working with this abtibody. We have a different anti-p53 antibody that works nicely and since our time as valuable as yours we cannot keep banging at a closed door. We would prefer refund. Can you credit the credit card that was used for the purchase of this antibody

ANSWER:

Thank you for your reply.
I am sorry that we were not able to resolve the problems that you were having with ab2433 in western blot. Therefore as you requested I am crediting the cost of the original purchase. Your credit note number is ******. The refund will be paid back onto the credit card that was used to make the purchase.
If there is anything else I can help you with, please let me know.

We have an answer to your inquiry:
Thank you for contacting Abcam.
I am sorry that you are having problems with ab2433, as you have opened and used the product you cannot return it, however under Abpromise we do guarantee that it will work in western blot on human samples. If it does not work as stated on the datasheet and we cannot determine why, then we can replace or refund the antibody.
Can you please provide me some more information about the nature of the problem and also some protocol information, to see if there is any suggestions I cna make that will resolve the problem.
1 - What sample type are you using?
Three human cell lines. Samples are the cell lysates. Cells were lysed in a lysis buffer with detergent (Triton X-100) for 20 min at 4 degrees, then centrifuged at 10.000 g for 15 min and mixed with 2x sample buffer for SDS PAGE.
2 - Are you seeing any bands, no bands etc?
We see nonspecific bands similar to bands seen after Ponseau staining.
3 - How much sample are you loading?
20 to 50 micrograms per lane.
4 - What type of blocking agent are you using?
5% BSA
5 - What primary antibody concentrations have you tried and how long do you incubate for?
1:200 dilution (1ug/ml) different incubation regimes: overnight at 4 degrees or 1 h at room temperature.
We do take all complaints on products seriously and we do our best to solve these problems.

ANSWER:

Thank you for your reply and for providing the information.
Comparing the information you provided to the information from the Abreviews and publications that have used this antibody before in human cells, the one protocol suggestion that I can make is to recommend using 5% milk rather than BSA as your blocking/incubation agent. I would also load 30ug of total protein and use a 1/500 primary antibody dilution with O/N incubation at 4C.
If after trying this protocol variation you are still having problems with the antibody, then as I mentioned previuosly I will be happy to either replace or refund the antibody as it is covered under our Abpromise.
Please let me know if there is anything else I can help you with.

Unfortunately, this antibody I purchased did not work in western blot. I used other controls that did work. Can I return it?

ANSWER:

Thank you for contacting Abcam.
I am sorry that you are having problems with ab2433, as you have opened and used the product you cannot return it, however under Abpromise we do guarantee that it will work in western blot on human samples. If it does not work as stated on the datasheet and we cannot determine why, then we can replace or refund the antibody.
Can you please provide me some more information about the nature of the problem and also some protocol information, to see if there is any suggestions I cna make that will resolve the problem.
1 - What sample type are you using?
2 - Are you seeing any bands, no bands etc?
3 - How much sample are you loading?
4 - What type of blocking agent are you using?
5 - What primary antibody concentrations have you tried and how long do you incubate for?
We do take all complaints on products seriously and we do our best to solve these problems.
I look forward to your reply.

LOT NUMBER lot No GR18443-8

ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Whole cell lysate from human post-mortem brain

PRIMARY ANTIBODY p53 Ab2433

DETECTION METHOD Scanning with Odyssey Infrared Imaging System

ANTIBODY STORAGE CONDITIONS +4

SAMPLE PREPARATION TX-Buffer and protein extracted using the trizol

AMOUNT OF PROTEIN LOADED 40 micrograms

ELECTROPHORESIS/GEL CONDITIONS Reducing and 12% gel

TRANSFER AND BLOCKING CONDITIONS Wet transfer/(over-night)1 h blocking in 5% milk solution in TBST

SECONDARY ANTIBODY Secondary antibody: IRDye 800CW Goat Anti-Rabbit IgG (H+L) Species: Anti- Rabbit Reacts against: Myc tag/ Beta-Actin Concentration or dilution: 1: 15000

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Changed the washing buffers from TBST to PBST and also reduced the blocking solution to 3%

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 this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. Reviewing this case, I would like to offer some suggestions to help optimise the results from ab2433. I would also appreciate if you can confirm some further details:

1. I can recommend to consider including a fresh positive control sample such as A-431 or HeLa lysate.

2. As far as I am aware, Trizol reagents are primarily designed for RNA or DNA isolation, and may not be optimal for good protein preparation. I can suggest to try RIPA lysis buffer if you have not already done so, which may provide a more suitable protein preparation.

3. Could you confirm has the transfer to the membrane and quality of the sample been assessed using a loading control?

4. Could you confirm the current vial of secondary antibody is working well with other antibodies?

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 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

I want to do a rat liver immunohistochemical staining of BCL2, p53, Ki67, Bax. Having no experience with it, I would ask you to tell me about the price, the primary and secondary antibodies, as well as all other reagents that I need to do this staining wich are compatibile with rat tissue and with each other. Thank you

ANSWER:

Thank you for your inquiry.

We have a number of antibodies in our catalog that might be of interest to you. I would like to direct you to the search facility on our website (www.abcam.com).

Please enter the name of the protein into the search box at the top of the home page. A list of products will be generated which can be further refined using the filters on the left hand side of the page. Using these filters will help you to identify products that have been tested in the applications and species you are interested in.

We have many BCl2 products in catalogue however I can recomend using ab18210 as it was tested in Immunohistochemistry on rat tissue sections.

ab18210: http://www.abcam.com/Bcl2-antibody-ab18210.html

The recomended p53 antibodies would be

ab26; http://www.abcam.com/p53-antibody-P-ab26.html ab28; http://www.abcam.com/p53-antibody-P-ab28.html ab2433; http://www.abcam.com/p53-antibody-ab2433.html

The recommended Ki67 antibodies are;

ab66155; http://www.abcam.com/Ki67-antibody-ab66155.html ab16667; http://www.abcam.com/Ki67-antibody-SP6-Proliferation-Marker-ab16667.html

We have region specific prices for the antibodies. The best way to know the price is by selecting the country of your origin displayed on top right corner of the datasheet. The system will automatically give you the price and shipping charges.

Please be also advised that you would also need fluorophore or enzyme conjugated secondary antibodies. I have attached a protocol booklet where protocol of each application has been nicely explained including the ingredients required. Please go through it and decide which antibody and reagent you require.

I hope this information is helpful to you. If you have specific questions about the products identified during the search, please do not hesitate to contact me.