Anti-Histone H3 (di+tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

I'm interesting to purchase antibodies against Histone 3 di methyl K4 (ab7766) or Histone H3 (di+tri methyl K4) ab6000, for the use in ChiP assay. The organism we are working is is Entamoeba histolytica and we know that the sequence around K4 is similar histones from other origins. But we do not know the degree of methylation (di or tri). I have few questions:

1. what is the price of each of the antibodies. 2. would you know any preference of using the di methyl which is polyclonal or the di+tri which is monoclonal for ChiP

in order to distinguish between transcriptional active and silenced genes.

ANSWER:

Thank you for your enquiry. I have performed an alignment of Entamoeba histolytica and homo sapiens and indeed there does seem to be some similarity in the amino acid sequence at lysine 4 of H3 (ARTK). These antisera have yet to be tested in this species. However this is not to say that it will not work, just that it has yet to be tested.

Entamoeba histolytica histone H3 (Q50XG0_ENTHI)

Entamoeba MARTKGHIERPSNKSAKAVKNVAFKAA Homo sapi -ARTK-QTARKSTGGKAPRKQLATKAA ****.: * *. . . *::* ***

ab7766 and ab6000 are £198 each and have both been tested using our own recommended ChIP protocol. This can be accessed at the following link:

http://www.abcam.com/index.html?pageconfig=view_protocol&pid=171

Both antisera work well by ChIP. However, in your example I would recommend determining the presence of either modification using mass spec before embarking on ChIP experiments as negative results are difficult to prove given that these antisera have not been tested on your species of interest.

I hope this information helps, please do not hesitate to contact me should you require further assistance.

Our group has purchased many antibodies from your company in the past, but one recent antibody I ordered did not work as it should have. This is ab6000, a mouse antibody to di/tri methylated histone H3 K4. This antibody should show exclusion from the inactive X chromosome in female cells, and it did not have this staining pattern. Therefore, I cannot use this antibody for experiments I have planned.

I would like to return this antibody for a refund. Please let me know how I can proceed.

batch# 46820

Answers to procedural questions are pasted below:

1. Please describe the problem (high background, no staining etc). High background

2. On what material are you testing the antibody in IHC? human female lung fibroblast WI-38 cells

3. How did you fix the samples? Paraformaldehyde

5. How did you block the unspecific binding sites?

blocking solution containing PBS, tween, goat serum, fish skin gelatin

6. Primary antibody Specification (in which species was it raised against)? peptide At what dilution(s) have you tested this antibody? 1:200, 1:500 Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)?

primary incubation time was overnight at 4 degrees. 4 five minute washes.These conditions were also used for a rabbit antibody to the same histone modification that works fine.

7. Secondary antibody What secondary antibody are you using? vector labs goat anti-mouse fitc

Specification (in which species was it raised against)? At what dilution(s) have you tested this antibody? 1:200

Incubation, wash steps? as above

Do you know whether the problems you are experiencing come from the secondary?

the secondary gives no signal when the experiment is performed in the absence of primary antibody

What detection method are you using? fluorescence

8. Background staining Please provide an image of your staining

diffuse nuclear staining -- no apparent exclusion from the inactive X chromosome

9. Which detection system did you use? fluorescent

10. Did you apply positive and negative controls along with the samples? Please specify.

Yes - These conditions were also used for a rabbit antibody to the same histone modification that works fine.

11. Optimization attempts How many times have you tried the IHC? twice

Do you obtain the same results every time? yes

What steps have you altered?

fixation conditions and antibody dilution

ANSWER:

Thank you for your e-mail and for providing further information about your IF protocol.

We are very sorry to hear that you are having problem with this antibody. We have sold many vial of this product and received good feedback from our customers.

Certainly we can offer you a reimbursement (either a credit note or a refund). We have two new batches in our stock and if you wish to try either of these, we would be more than happy to send you one vial for testing.

Please do let us know how you would prefer to proceed. We look forward to hearing from you soon.

Thanks for your answer.

This ist our customers reply:

Please realize I am working with Drosophila polytene chromosomes and not with mammalian tissues! 1. In my experiment I did not stain a whole tissue, but chromosomes that were released from nuclei by a squashing procedure (standard for polytene chromosomes of Drosophila !). Therefore, access of the Ab to the nucleus is not a problem, as the nuclei are disrupted and chromosomes are free on the slide. Inclusion of Triton for the reason to gain access to the antigene is therefore useless.

2. The Ab was not exposed to the fixative acetic acid/ formaldehyde, as chromosomes are rehydrated in PBS after fixation. The incubation of the Ab with chromosomes is in PBS over night. This is a standard procedure, very simple and successfully used for many decades now, with several thousand Ab in as many different Drosophila laboratories. When the antigene on the histones is not accessible to the monoclonal Ab this might be due to the chromosomal environment. A polyclonal serum will certainly give a different result.

ANSWER:

Thank you for getting back to us and for providing further details to our questions.

We are very sorry that your customer is still struggling with this antibody; we have very good feedback about it from our customers.

We can offer either a reimbursement for your troubled customer or another product from our catalogue.

Please do let us know how he/she would like to proceed. We look forward to hearing from you soon.

BATCH NUMBER 93153

DESCRIPTION OF THE PROBLEM No signal above background

SAMPLE Drosophila melanogaster wild type polytene chromosomes

PRIMARY ANTIBODY Ab6000 / mouse monoclonal / 1:100 in PBS / incubation 16 h RT / Washing of slides in 80 ml PBS for 10 min, twice

SECONDARY ANTIBODY Rhodamine conjugated goat-anti-mouse, Dianova, 1:1000, incubated For 1 h at RT, washed in 80 ml PBS for 10 min

DETECTION METHOD Zeiss epifluorescent microscop

POSITIVE AND NEGATIVE CONTROLS USED Standard procedure for chromosomal staining with numerous Ab In our lab

ANTIBODY STORAGE CONDITIONS 4?C

FIXATION OF SAMPLE 5 min 1% Formaldehyde/50% acetic acid, 10 min 100% Ethanol 10 min rehydration in PBS

ANTIGEN RETRIEVAL Mechanically disrupted salivary glands and squashed chromosomes

BLOCKING CONDITIONS none

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

ADDITIONAL NOTES the customer wants to try another antibody. For AB6000 he wouls like a refund! Thanks

ANSWER:

Thank you for your enquiry and for completing the on-line Questionnaire. We have received very good feedback about this product from our customers.

1. As the datasheet indicates this antibody has been tested and characterised for IF studies. The cells were fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour. Permeabilization is a very important step since this target is nuclear protein.

2. One of our customers has submitted us a review in which he used 4%PFA plus 0.5% glutaraldehyde as fixatives.

As we understand form the e-mail, your troubled customer has used 1% Formaldehyde/50% acetic acid. We have not tested this type of fixative and it is possible that the 50% acetic acid may be too strong for this particular antibody. We would suggest modifying the fixation step if it is possible and applying permeabilization.

Should you need further help, please do not hesitate to contact us again.