Anti-Nrf2 antibody (ab53019)

I would like to test ab53019 for use in IP.

ANSWER:

DISCOUNT CODE: ***
Expiration date: September 15th, 2012

I am very pleased to hear you would like to accept our offer and test ab53019 inIP. This code will give you: 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for IP 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: http://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: http://www.abcam.com/collaborationdiscount.

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Nuclear and cytoplasmatic exctract of endothelial cells (EA.HY926, cell line commercial) PRIMARY ANTIBODY Anti-Nrf2 antibody (ab54364) monoclonal mouse diluent used was 0.3% Non-Fat Dry Milk TBS-T 0.1% (1X) dilutions were tested 1:1000 1:500 1:200. Incubation time was overnight at 4°C in agitation. Wash step was 3 times with TBS-T 0.1% (1X) for 15 min. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED NO ANTIBODY STORAGE CONDITIONS Antibody is storage at -20°C SAMPLE PREPARATION we used cytoplasmatic lysis buffer ( 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.3% Triton 100X, protease inhibitor cocktail, 0.5 mM PMSF, 1 mM NaVO3, 5 mM -glycerophosphate) nuclear lysis buffer (20 mM HEPES, 1.5 mM MgCl2, 0.6 mM KCl, , 20% glycerol, 0.2 mM EDTA, protease inhibitor cocktail, 0.5 mM PMSF, 1 mM NaVO3, 5 mM -glycerophosphate) and sample Buffer 5X (60 mM Tris HCl, 25% glycerol, 2% SDS, 5% -mercaptoethanol, 0.1% bromophenol). Heating sample was 95°C at Thermomixer for 5 min. AMOUNT OF PROTEIN LOADED 30 ug of protein ELECTROPHORESIS/GEL CONDITIONS Reducing gel, 10% of the SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Transfer period was 1 hour blocking agent was 3% Non-Fat Dry Milk TBS-T 0.1% (1X) SECONDARY ANTIBODY Goat anti-mouse IgG-HRP conjugate diluent used was 0.3% Non-Fat Dry Milk TBS-T 0.1% (1X) dilution used was 1:5000. Incubation time was 1 hour at temperature room in agitation. Wash step was 3 times with TBS-T 0.1% (1X) for 15 min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We altered only primary antibody dilutions (1:1000 1:500 1:200) ADDITIONAL NOTES First time we used another primary antibody (ab 53019) anti-Nrf2. This primary antibody was perfect to detected Nrf2 in our samples. For this reason we ask if you can change primary antibody ab 54364 with primary antibody ab 53019.

ANSWER:

Thank you again for contacting us and letting us know about this trouble.

We are sending a vial of ab53019 on the order ***.

Please let me know if you have any questions or if there is anything else that we can do for you.

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Nuclear and cytoplasmatic exctract of endothelial cells (EA.HY926, cell line commercial) PRIMARY ANTIBODY Anti-Nrf2 antibody (ab54364) monoclonal mouse diluent used was 0.3% Non-Fat Dry Milk TBS-T 0.1% (1X) dilutions were tested 1:1000 1:500 1:200. Incubation time was overnight at 4°C in agitation. Wash step was 3 times with TBS-T 0.1% (1X) for 15 min. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED NO ANTIBODY STORAGE CONDITIONS Antibody is storage at -20°C SAMPLE PREPARATION we used cytoplasmatic lysis buffer ( 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.3% Triton 100X, protease inhibitor cocktail, 0.5 mM PMSF, 1 mM NaVO3, 5 mM -glycerophosphate) nuclear lysis buffer (20 mM HEPES, 1.5 mM MgCl2, 0.6 mM KCl, , 20% glycerol, 0.2 mM EDTA, protease inhibitor cocktail, 0.5 mM PMSF, 1 mM NaVO3, 5 mM -glycerophosphate) and sample Buffer 5X (60 mM Tris HCl, 25% glycerol, 2% SDS, 5% -mercaptoethanol, 0.1% bromophenol). Heating sample was 95°C at Thermomixer for 5 min. AMOUNT OF PROTEIN LOADED 30 ug of protein ELECTROPHORESIS/GEL CONDITIONS Reducing gel, 10% of the SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Transfer period was 1 hour blocking agent was 3% Non-Fat Dry Milk TBS-T 0.1% (1X) SECONDARY ANTIBODY Goat anti-mouse IgG-HRP conjugate diluent used was 0.3% Non-Fat Dry Milk TBS-T 0.1% (1X) dilution used was 1:5000. Incubation time was 1 hour at temperature room in agitation. Wash step was 3 times with TBS-T 0.1% (1X) for 15 min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? We altered only primary antibody dilutions (1:1000 1:500 1:200) ADDITIONAL NOTES First time we used another primary antibody (ab 53019) anti-Nrf2. This primary antibody was perfect to detected Nrf2 in our samples. For this reason we ask if you can change primary antibody ab 54364 with primary antibody ab 53019.

ANSWER:

Thank you for contacting us and for letting us know about the trouble with ab54364. I am sorry that this antibody did not work as expected.

Wewill be happy to send a free of charge vial of ab53019 in exchange for ab54364. I have asked my colleagues to send this to you, and we will update you shortly with the order number.

Please let me know if you have any questions or if there is anything else that we can do for you.

I am very interested in one of your antibodies, specifically ab53019 (anti-Nrf2). According to my sequence analysis it would be suitable for the detection of a homologous protein in the Cnidarian Hydra. It is very difficult (and expensive) for us to actually find antibodies that bind also to Hydra epitopes. Would it be possible to receive a small aliquot for testing the antibody on condition that I will provide data of my results?

Best regards,

ANSWER:

Thank you for contacting Abcam.

We do not provide sample sizes of our antibodies as under our Abpromise we guarantee our products to work as stated on the datasheet.

If however a customer, like yourself, wishes to test an antibody in an untested species, then as long as the sequence homology between the immunogen and untested species is 85% or greater, then you can take advantage of our testing discount program (see link below):

http://www.abcam.com/index.html?pageconfig=resource&rid=11998



If you could please send me your sequence for Cnidarian Hydra, I can do an alignment and see if you would be eligible for our testing discount program.

I look forward to your reply and if there is anything else I can help you with, please let me know.

Incorrect bands in Western blot with mouse knockout and wild type liver samples. Previous vial worked better.

ANSWER:

Thanks for your call today, and I apologize for all the trouble with these Nrf2 antibodies. As we discussed, I am sending a vial of ab92946 on the order ***, which should arrive tomorow. Please keep me updated about the results using this antibody. If you would like to try any of our antibodies (Nrf2 or other targets) in a species that is not listed in the "Reacts With" section of the datasheet, please let me know and I will be happy to arrange our testing discount program for you. I hope this information is useful, but please let me know if you need anything else and I'll be happy to help you. Have a great week!