Anti-Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

I'm using these antibodies to detect histone modifications of histone extractions of 293T cells and not seeing any signal.

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 a free of charge replacement. 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.  

Our lab bought several antibodies from your company before. Now I am trying to do ChIP and particularly interested in trying the PCR primers you guys used. I notice that the website of your company does not list this information whereas the delivered information sheets do. I am wondering whether you can help me with the sequences of PCR primers for the following antibodies: ab8895, ab8580, ab2886, ab3594, and ab2621. I appreciate if you can mail me information sheets regarding these A/bs.

ANSWER:

Thank you for your enquiry.

All antibodies that are batch tested here at Abcam have the primers that were employed during the testing published as a separate page which can be accessed as a clickable link on the antibody datasheet. For example on the antibody datasheet for ab8895 under "specific protocols" there is a clickable link;

"PCR primers used to test ab8895 in ChIP at Abcam"

Here you will find all the information necessary to test the antibody. You can locate the same PCR primer information on each of the antibody datasheets you refer to.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

please find in the following a complaint for 2 vials of ab2886:

Histone H3 (mono methyl K79) antibody, rabbit polyclonal 1. Order details: ? Product code: ab2886 ? Batch number: 90959 ? Abcam order or Purchase order number: ? Antibody storage conditions (temperature/reconstitution etc): in ?80C freezer until use.

2. Please describe the problem (high background, wrong band size, more bands, no band etc). We are unable to obtain any signal (either specific or non-specific) using this antibody. However, in general we can detect histones very well on Western blot (for example using the Abcam antibodies against H3 dimethyl and trimethyl K79 on the same lysates); our problem is specific for this antibody.

3. On what material are you testing the antibody in WB? ? Species: Yeast ? Cell extract or Nuclear extract: Cell extract ? Purified protein or Recombinant protein: endogenous protein

3. The lysate ? How much protein was loaded: 3 ug, 5 ug or 7 ug of histone H3 ? What lysis buffer was used: 1% SDS, 8M urea, 10 mM MOPS pH 6.8, 10 mM EDTA, 1mM DTT. ? What protease inhibitors were used: PMSF + benzamidine + pepstatin + leupeptin ? What loading buffer was used: same as lysis buffer ? Did you heat the samples: temperature and time: 65 C, 10 min

4. Electrophoresis/Gel conditions/ Transfer conditions ? Reducing or non reducing gel: reducing ? Gel percentage : 15% ? Transfer conditions: semi dry transfer 5. Blocking conditions ? Buffer: TBST ? Blocking agent: milk, BSA, serum, what percentage: I tried 5% milk, 3% BSA and 5% milk with an extract fom a dot1 knockout (the enzyme that performs H3K79 methylation). ? Incubation time: 1 hr ? Incubation temperature: room temp

6. Primary Antibody ? Specification (in which species was it raised against): rabbit ? At what dilution(s) have you tested this antibody: 1:500 as advised by Abcam. ? What dilution buffer was used: 2% milk in TBST ? Incubation time: 16 hrs ? Incubation temperature: 4C ? What washing steps were done: 3x 5 mins TBST

7. Secondary Antibody ? Specification (in which species was it raised against)? Donkey anti-rabbit ? At what dilution(s) have you tested this antibody: 1:5000 ? Incubation time: 1 hr, room temp ? Wash steps: 3X 10 mins ? Do you know whether the problems you are experiencing come from the secondary? Unlikely, because all other rabbit antibodies we use give good signals when combined with this 2nd antibody.

8. Detection method ECl, ECl+, other detection method: ECL (Amersham) and I tried SuperSignal a couple of times for extended duration chemoluminescence.

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

10. Did you apply positive and negative controls along with the samples? Please specify. I always use a lysate from a dot1 knockout strain which does not have any H3K79 methylation and one from a dot1 wild type strain which does. I also tried older lysates which gave a nice signal with the old batch of H3K79-Me1 antibody (these were also not detectable).

11. Optimization attempts ? How many times have you tried the Western? 8x (by two different people) ? Do you obtain the same results every time e.g. are background bands always in the same place? Yes, we never detect any signal. ? What steps have you altered? Blocking, detection method, amount of lysate loaded.

Best Regards BIOZOL Diagnostica Vertrieb GmbH Ralf

von/from Dr. Ralf Baeuerle BIOZOL Diagnostica Vertrieb GmbH Postfach/P.O. Box 2022 D-85386 Eching Germany Fon +49 (0)89 3799 666-6 Fax +49 (0)89 3799 666-99 e-mail r.baeuerle@biozol.com oder/or r.baeuerle@biozol.de Website http://www.biozol.com oder/or http://www.biozol.de

ANSWER:

Thank you for your enquiry and for completing our technical questionnaire so comprehensively.

I am sorry to hear that you have been having difficulties with this antibody.

It is necessary to apply our mono methyl K79 antibody, ab2886 at a higher concentration than that of our di and tri methyl K79 antisera. I have consulted the lab and I would like to recommend that the antibody is applied at a higher concentration than 1:500. (e.g. 1:100) I appreciate that this is the concentration stipulated on our datasheet. However, we do observe occasional batch to batch variation of the efficiency of some of our antibodies. It may well be that the batch that you are using requires a slightly higher concentration.

It may also be useful to use a histone extraction rather than a cell extract as this will serve to concentrate the epitope presented to the antibody. Again I appreciate that the conditions that you have been using have been successfully applied using two of our alternative methyl K79 antibodies, but I feel that these suggestions will fully determine whether it is simply the detection threshold of the antibody or whether the antibody has been compromised in some way.

For an excellent yeast histone extraction protocol please see the following link from the Grunstein lab.

http://mgwww.mbi.ucla.edu/modules/wiwimod/index.php?page=Yeast+Histone+Prep&back=Protocols

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.