Anti-NQO1 antibody [A180] (ab28947)

- Cell type and name e.g. human hela cells = mouse embryonic fibroblast from wild-type and Nrf2 knockout mice
- Lysis buffer used = RIPA buffer (Sigma) with complete mini tablet (Roche)
- Amount of lysates loaded = 50ug
- Are the cell lysates used or IP lysates?
- Dilution of primary and secondary antibody = primary, 1:2500. secondary, 1:3000
- Dilution and species of secondary antibody = goat antimouse (SantaCruz, since Abcam 2nd antibody is not good as SantaCruz), 1:3000
- Protease inhibitors used = no protease inhibitor was used since it's all included in complete mini tablet
- Info of positive control = sulforaphane (ReviewDirect and indirect antioxidant properties of inducersof cytoprotective proteins byAlbena T. Dinkova-Kostova and Paul Talalayhttp://onlinelibrary.wiley.com/doi/10.1002/mnfr.200700195/abstract



- Purchase order number = not sure since I take 10ul from other lab in UKM
- Preared fresh lysates or used stored lysates = stored lysates but aliquote into 3 tubes. this tube is first came out from -80.



For yourinformation, I used Abcam heme oxygenase with same sample protein but there are no problem at all. Regards,

ANSWER:

Thank you very much for your email.

The antibody should have worked with the described conditions we are unsure about the cause of band at 50 kDa. There could be one or 2 reasons reason

- The stored lysates are used; I would recommend using fresh lysates. In many cases the cause of multiple bands is improperly stored or long stored lysates.
- Heavy chain of antibody which is 50 kDa of molecular weight which gives band If the same antibody is used for IP and western blot. This does not seem the case because you are not using IP lysates.

In the following publication author have used UV radiation treated human fibroblast got a NQO1 bands. http://cancerres.aacrjournals.org/content/66/17/8421.full. This publication may provide further insight in your research.

Try using fresh lysates, 1/1000 antibody dilution or UV irradiated cell lysates.

We have not received any complaint for this lot so I am sure results will improve following these suggestions.

Please be also advised that, as you have received this antibody as a gift from other lab so we will only provide troubleshooting help.

I hope these suggestions will help to improve results. Should you have any other question please do not hesitate to ask.

As you can see from the enclosed images, she had a faint band (abt 30kDa) and a very strong band at around 50 kD. She was very puzzled about this strong 2nd band at 50 kD as she did not see this when using same anti-NQO1 from another manufacturer…. She is also afraid to dilute further as she might “lose” the true band on 30 kD….
I just rightly assume all her protocols are optimized as her group has been working on WB experiments for 2 years now & they have been purchasing some Abcam antibodies which had gave excellent results thus far…
Is there any explanation that I could provide to her? Please advise.
Thanking you in advance! Best regards,

ANSWER:

Thank you for contacting us. I am sorry to hear about the wrong band size.

Could you please provide theinformationwhich will help us to understand the cause of wrong band size. The info we need is

- Cell type and name e.g. human hela cells
- Lysis buffer used
- Amount of lysates loaded
- Are the cell lysates used or IP lysates?
- Dilution of primary and secondary antibody
- Dilution and species of secondary antibody
- Protease inhibitors used
- Info of positive control
- Purchase order number
- Preared fresh lysates or used stored lysates

Looking forward to hearing from you soon.

Hi,
Can I know why this antibody MW shows at 50 kDa on our blot instead of 30 kDa as stated in the data sheet. Please advise.
Thank you

ANSWER:

Thank you for taking the time to contact us. I am sorry to hear you have some concerns regarding the results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for the applications and species listed on the datasheet. 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.

Reviewing the details, we would expect a band at 30 kDa and not 50 kDa. Therefore, 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,including molecular weight markers,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.

I would like to test ab28947 anti-NQO1 in FFPE monkey tissue.

ANSWER:

I am very pleased to hear you would like to accept our offer and test ab28947 in monkey. This code will give you 1 freeprimary antibodybefore the expiration date. To redeem this offer, please submit an Abreview for monkey 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.