Anti-c-Myc antibody [9E11] - ChIP Grade (ab56)

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Inquiry: We used the above antibody in our Chip experiments in S.cerevisiae after our last antibody (Santa Cruz Sc-40) stoped working. We prepare whole cell lysates and following the abcam protocol booklet we found no enrichment of our protein/s of interest at known promoters by qPCR. (Swi4-myc at SVS1, CLN2 etc) We tested our strains for the presence of myc-tag by western and the tag was present. We tried increasing amounts of antibody up to 7.2µg per IP. Whole cell extract gave readings on qPCR, and we also IP'd with anti H3K9ac as a positive control. Our buffers are the same as recommended in the abcam protocols booklet. How do we go about aquiring another lot of this antibody or getting a refund?

ANSWER:

Thank you for taking time to contact us with these details. I am sorry to hear that this antibody is not providing satisfactory results.
We firstly would appreciate to receive more details on the experimental procedure. I have therefore attached a questionnaire to this email, which should not take you more than 5 to 10 minutes to fill. These details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality as well as to provide troubleshooting suggestions if adequat. Thank you for your cooperation!
Can you please also confirm whether you have tested if the ab56 can IP the myc tagged protein or not?
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I am looking forward to hear back from you.

I'd like to know whether this antibody works efficiently in ChIP (conventional paraformaldehyde fixation) of Myc-tagged (one tag) proteins expressed in mammalian tissue culture cells. .

ANSWER:

Thank you for your enquiry.

This antibody was qualified as "ChIP grade" following its application in the following publications;

Robert F et al. Global position and recruitment of HATs and HDACs in the yeast genome. Mol Cell 16:199-209 (2004). PubMed: 15494307

Zhang H et al. The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres. Mol Cell Biol 24:9424-36 (2004). PubMed: 15485911

We have received reports suggesting that the antibody does not give a huge enrichment but that the enrichment is specific. Furthermore it is guaranteed for this purpose. I would recommend that you consult the publications for details of the approach that was adopted. From my recollection they did use proteins tagged with more than one Myc-tag.

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

I am thinking of doing ChIP on a transcription factor in live insects (Drosophila melanogaster). I am not sure which tag would be best for this purpose. I am thinking about fusing my protein to 5 tandem myc tags or HA tags using your anti-myc or anti-HA antibodies for the ChIP. Could you guys provide me with some advice as to the best tags for doing ChIP on Drosophila? Thanks.

ANSWER:

Thank you for your enquiry.

At Abcam we have validated a panel of epitope tagged antibodies for use in chromatin immunoprecipitation (see link at the bottom of this email). I would like to recommend the following antibodies in view of the tags that you are proposing.

ab9132 Goat polyclonal to Myc tag ab9110 Rabbit polyclonal to HA Tag

Both were validated and have been shown to work very well by ChIP. Indeed ab9110 is a very good seller. Furthermore we guarantee them both for this purpose.

http://www.abcam.com/index.html?pageconfig=resource&rid=10595&pid=5

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

I am a postdoc at University of Texas at Austin, and I am currently trying toperform the chIP experiments using c-Myc anitbody to detect c-myc binding sites in human cell lines such as hela cells. we have tried Abcam's chIP gradeantibody (ab56), then did qPCR to evaluate the efficency of antibody, but chIP DNA vs input DNA only give 2 or 3 fold enrichment for a known c-myc bindingsites (promoter region of gene CAD).I am wondering if the enrichment is right, do you have any reference can be as apositive control to evaluate the efficency of chIP for c-Myc.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. This antibody has been qualified as suitable for chromatin immunoprecipitation due to its application in publications that have applied it using chromatin immunoprecipitation. In particular;

Zhang H et al. The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres. Mol Cell Biol 24:9424-36 (2004). PubMed: 15485911

However, from examination of this publication I can see that this antibody was employed to target Sin3 tagged with multiple copies of the Myc epitope. Therefore the enrichment that was detected in this publication is unlikely to reflect the enrichment when targeting endogenous Myc given multiple epitopes and transient expression.

You may wish to consult the recent publication Guccione et al., 2006 Myc-binding-site recognition in the human genome is determined by chromatin context. Nat Cell Biol 8:764-70 for positive control loci and conditions suitable for targeting Myc in ChIP experiments. In this publication ChIP was performed as detailed in Fernandez, P. et al. Genomic targets of the human c-Myc protein. Genes Dev 17, 1115–1129 (2003).

Given poor enrichment you may wish to consider increasing the duration of formaldehyde fixation and/or combining reduced stringency washes.

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

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 139279

DESCRIPTION OF THE PROBLEM mulitple bands, very high background

SAMPLE MCF7 and T47D cell lysates

PRIMARY ANTIBODY Used Abcam c-myc (cat. # ab56) diluted 1:250, 1:500, and 1:1000 in 5% milk. Incubated for 1 hour and washed with 1X TBST buffer three times (10 minutes per wash).

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Tested beta-Estradiol stimulation versus horomone depletion. Also compared RNAi knockdown to normal expression.

ANTIBODY STORAGE CONDITIONS -20 - -30 degrees

SAMPLE PREPARATION Lysis Buffer with PI cocktail solution and PMSF. Added SDS loading buffer and boiled smaple for a minimum of 5 minutes.

AMOUNT OF PROTEIN LOADED 80ug

ELECTROPHORESIS/GEL CONDITIONS 10% SDS PAGE Gel

TRANSFER AND BLOCKING CONDITIONS Transfer @ 50 volts overnight. Blocking with 5% milk.

SECONDARY ANTIBODY BioRad Goat-Anti-Mouse was used as a secondary, diluted 1:2000 in 5% milk. Incubated for 1 hour and washed with 1X TBST buffer three times (10 minutes per wash).

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Tested different dilution of primary antibody, varied tranfer time from 90V for at least 4 hours to 50V overnight, and varied blocking time from 1 hour to overnight.

ANSWER:

Thank you for your enquiry.

I am sorry for the confusion. I made a mistake of sending you a response intended for another customer.

I am sorry to hear that you have been having difficulties with c-Myc antibody [9E11] - ChIP Grade (ab56). This antibody is a good selling antibody that has received a favourable Abreview from one of our customers. Although I acknowledge that she did observe a degree of non-specificity with additional bands at 75KDa, 110KDa and 140kDa.

At this stage I would like to recommend that you reduce the mass of protein that you are loading on the gel and perform an overnight incubation at 4oC using the antibody at a high dilution. We recommend 20-30ug of protein.

In view of the background that you have been observing I would also like to recommend that you try changing the blocking agent that you have been using. This often leads to a cleaner more specific blot. I would like to recommend that you use 3% BSA rather than non-fat milk.

Increasing the number of wash steps after the primary antibody incubation is also a good way to address the high background that you have been observing.

Please do not hesitate to contact me should these suggestions not lead to an improvement in your results.