Anti-NFkB p105 / p50 antibody - ChIP Grade (ab7971)

Dear Sir or Madam,

As we are starting a new project involving the NFkB pathway we are interested in several of your antibodies, mainly in the following order numbers: ab52175, ab7547, ab7971, ab31409
Since we do not know if the antibodies are suitable for our project we would like to test them before buying a larger amount. Could you make us an offer for a test kit with the above mentioned antibodies? We would also appreciate if you could suggest additionally antibodies that could be used in western blot as well as immunofluorescence for the NFkB pathway in mouse and human.

Thank you in advance.

Best regards,

ANSWER:

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our testing discount program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

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I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

I am interested in using anti NFkB –p50 antibody, ab 7971 but before ordering I would like to confirm few technical details. My construct will encode NFkB-50 from AA 40 to AA 366 (40-366), I would like to know whether your antibody is suitable to perform WB for the expressed protein? Kindly confirm

ANSWER:

Thank you for your enquiry and your interest in our products. This is to confirm that the immunogen sequence of this antibody was a synthetic peptide derived from the region 330aa to 433aa of the human NFkB p50 subunit. Does your construct contain the sequence of human protein? Are you going to use any tagging which may interact with the recognition of the epitope(s)? I hope this helps and if I can assist further, please do not hesitate to contact me.

Thank you so much. The PO numbers are xxx for the vial ordered late november and yyyy ordered mid october.

ANSWER:

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued 2 free of charge replacements to replace the 2 vials of ab7971. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

1. Order details: 1 Batch number: NFkB p105 / p50 antibody - ChIP Grade (ab7971) 2 Abcam order or Purchase order number:S060606 3 Antibody storage conditions (temperature/reconstitution etc): 4? without reconstitution

2. Please describe the problem (high background, wrong band size, more bands, no band etc). Multiple bands (3 bands) located 50 kDa

3. On what material are you testing the antibody in WB? * Species: Brain tissue of SD rats * Cell extract or Nuclear extract: Nuclear extract

3. The lysate 1 How much protein was loaded : 50ug 2 What lysis buffer was used: What protease inhibitors were used:

After the rats were killed, the brains were rapidly removed and the LA and BLA were dissected out. The tissues were ground using a Dounce grinder with a loose pestle in iced-chilled buffer (15 mM HEPES, 60 mM KCl, 1 mM NaCl, 0.25 M sucrose, 5 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 15 µg/ml leupeptin, 2 mM NaF, and 1 mM sodium orthovanadate). The homogenate was centrifuged for 10 min at 2000 rpm, and the pellet was resuspended in buffer (10 mM HEPES, pH 7.2, 15 mM MgCl2, 10 mM KCl, 1 mM PMSF, 2 mM NaF, 15 µg/ml leupeptin, and 1 mM sodium orthovanadate). After a brief vortex, they were incubated on ice for 10 min and lysed with a tight pestle. The homogenate was centrifuged at 4000 rpm for 10 min. The pelleted nuclei were resuspended in 40 to 60 µl of extraction buffer consisting of 100 mM HEPES, pH 7.2, 1.5 mM MgCl2, 1 mM EDTA, 0.8 M NaCl, 15% glycerol, 2 mM NaF, 1 mM PMSF, 15 µg/ml leupeptin, and 1 mM sodium orthovanadate and were incubated on ice for 2 to 4 h. The nuclear suspension was centrifuged at 14,000 rpm for 30 min at 4°C, and the supernatant was saved.

Mol Pharmacol 65:1286-1292, 2004

2 What loading buffer was used: see attached 3 Did you heat the samples: temperature and time: 95?, 5min

4. Electrophoresis/Gel conditions/ Transfer conditions 2 Reducing or non reducing gel: 3 Gel percentage : 10% 4 Transfer conditions: wet transfer(Bio-Red), 300mA , 1h

5. Blocking conditions 1 Buffer: TBS 2 Blocking agent: milk, BSA, serum, what percentage: 5% non-fatty milk 3 Incubation time: 1h 4 Incubation temperature: 25?

6. Primary Antibody

1 Specification (in which species was it raised against): rat * At what dilution(s) have you tested this antibody:?1:1000??1:3000??1:5000?in 3% BSA * What dilution buffer was used: TBS * Incubation time: at 4°C with gentle shaking, overnight. * Incubation temperature: 4°C * What washing steps were done: TBST(1X TBS, 0.1% Tween-20), 10 mins and 3 times

7. Secondary Antibody 2 Specification (in which species was it raised against)? Rabbit [another company] 3 At what dilution(s) have you tested this antibody: ?1:3000? 4 Incubation time: RT 1h 5 Wash steps: TBST(1X TBS, 0.1% Tween-20), 10 mins and 3 times 6 Do you know whether the problems you are experiencing come from the secondary? never

8. Detection method ECl, ECl+, other detection method: ECl (PerkinElmer :NEL105)

9. Background bands * Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes * Is the blocking step sufficient? Yes * Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes * At what size are the bands migrating? Could they be degradation products of your target? NO. The bands are almost the same size. * Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

11. Did you apply positive and negative controls along with the samples? Please specify. NO

10. Optimization attempts * How many times have you tried the Western? 5 times * Do you obtain the same results every time e.g. are background bands always in the same place? Yes. I always obtained 3 bands in 50kDa. * What steps have you altered? Antibody titer

ANSWER:

I'm sorry to hear one of your customers is experiencing a problem with ab7971. I think the customer's protocol is mostly very good and similar to the one I use, however I think there are three suggestions I would like to make:

1) please check that the customer uses a reducing gel and has boiled his samples in the loading buffer. I think it is likely he has done so but it is not clear on the form so I think it is worth double checking this is the case.

2) mixing blocking agents can give non specific bands. Please try two conditions: 5% milk 1hr and on a different membrane 5% BSA. I typically find BSA gives better results but this depends on the antibody. Please incubate the antibody in TBST only.

3) the membrane looks overexposed. I would recommend to expose less and see if the 50kda band only is seen, and to also try to decrease the amount of antibody, trying 1:10000 or more.

I hope these recommendations will help the customer. If he still experiences problems please do not hesitate to let me know and I can offer a replacement vial or refund,

ANTIBODY CODE ab7971 BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I had a band size of 50 kDa, and another around 65 kDa.

SAMPLE Samples were form primay astrocyte cultures form neonatal mouse brain.

PRIMARY ANTIBODY 1:200 diltuion, abcam

SECONDARY ANTIBODY 1:1000 dilution, Amersham, anti-Rabbit HRP

DETECTION METHOD Pierce

ANTIBODY STORAGE CONDITIONS 4C

SAMPLE PREPARATION I used lysis buffer with protease inhbibitors.

AMOUNT OF PROTEIN LOADED 30 ug

ELECTROPHORESIS/GEL CONDITIONS 8% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS 5% blocking agaent/PBS+ 0.05% Tween-20

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

ADDITIONAL NOTES I would like to know what is the exact size for NFkB p50 on Western, done in your lab?It says in the data sheet that it is a polyclonal Ab, and would detect p105 and p50... Didn't you have any non specific bands? Do you have any data on the size of NFkB p50 from mouse?

ANSWER:

I'm sorry to hear that you are experiencing problems with this antibody. I have enquired with the originator of this antibody and they have said that they experienced no problems with non-specific bands. In mouse the antibody will detect a p50 and p105 band (the bands are 50 kDa and 105 kDa) in NIH/3T3 whole cell lysates. I suggest that you run a positive control, such as NIH/3T3 whole cell lysates or K-562 and A-431 whole cell lysates. If you have any more questions, please contact us again.