Anti-HDAC2 antibody - ChIP Grade (ab7029)

BATCH NUMBER 180177 ORDER NUMBER 1693599

X-CHIP or N-CHIP How many volume use for ChIP or tell me how concentration of HDAC2 for this vial (xxmg/ml). Thank you!

ANSWER:

Further to your previous enquiry.

The protein concentration of ab7029 lot number 180177 is 12.4mg/ml (as determined by UV absorbance).

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

BATCH NUMBER 123137 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM multiple bands on IP for serine or threonine phosphorylation antibodies (ab6626, ab2286)- dont know which one is HDAC-2 as reprobing with HDAC-2 ab (ab7029 - one which I immunoprecipetated with) gives 2 and, the strong of which is not at the correct molecular weight & I get 2 bands (on the phospho blots) that are on/just abouve 50kDa - are any of these bands on the phospho blots HDAC-2?? and which (if any) on the native HDAC-2 blots is HDAC-2?? Also please could you recomend at positive control lysate to use to establish the band - Hela?????

SAMPLE nuclear extract from U937 cells (human)

PRIMARY ANTIBODY HDAC-2 ab7029 IP: 1/2500 dilution for 1 hour at 4 degrees with agitation WB: 1/800 dilution for 1.5 hour with 1%BSA-TBS-Tween(0.1%) with agitation washing - 3x7 min with TBS-Tween(0.1%)

WB Serine Phosphorylation - ab6629 - 1/300 dilution in 1%BSA-TBS-Tween(0.1%) for 1.5 hours at room temperature WB Threonine phosphorylation - ab2286 - 1/600 dilution (conditionjs same as for serine)

DETECTION METHOD Used ECF (Amersham) and analysed on phosphoimager

POSITIVE AND NEGATIVE CONTROLS USED no positive/negative controls

ANTIBODY STORAGE CONDITIONS aliqouted (5ul) and stored at -20

SAMPLE PREPARATION Nuclear Extract All steps to be done on ice or at 4?C & add Phosphates Inhibitor cocktail (Calbiochem) just before us.

3. Re-suspend in 500?l Lysis buffer in 1.5ml eppindorf tube 4. Incubate on ice for 10 min 5. Vortex samples then centrifuge at 14000rpm for 5 min (4?C) 6. Remove supernatant (cytoplasic fraction) store at -80?C 7. Re-suspend pellet in 500?L Wash buffer 8. Vortex or brush against eppindorf rack 9. Centrifuge at14000rpm for 5 min (4?C) 10. Discard all supernatant 11. Re-suspend pellet in Buffer B (volume depends on No. of cells) 12. Vortex/brush the eppindorf?s gainst the eppindorf rack 13. Incubate on ice for 20 min 14. Vortex/brush the eppindorf?s against the eppindorf rack 15. Centrifuge samples at 14000rpm for 5min 16. Transfer supernatant into fresh eppindorfs 17. Add Buffer C (volume depends on No. of cells) 18. Aliquot & store at -80?C

Buffers

Buffer A: Final Concentratio 10mM HEPES pH7.9 1.5mM MgCl2 10mM KCl *0.5% NP40 1mM DTT 1M Protein Inhibitor tablet dH2O 27.78ml

Just before use: Phosphatase Inhibitors 1/100 dilution

Wash Buffer Final Concentration 10mM Tris HCl pH 7.4 13mM EDTA 0.5M dH2O 4820 Protein Inhibitor tablet

Buffer B: Final Concentration 20mM HEPES pH 7.9 1.5mM MgCl2 0.42 mM NaCl 0.2mM ETDA 25% Glycerol 1mM DTT 1M Protein Inhibitor tablet dH2O 12.83ml

Just before use: Phosphatase Inhibitors 1/100 dilution

Buffer C: Final Concentration 20mM HEPES pH 7.9 50mM KCL 0.2mM EDTA 1mM DTT 1M Protein Inhibitor tablet dH2O 18.57ml

Just before use: Phosphatase Inhibitors 1/100 dilution * Protein Inhibitor tablets ? Roche ***for IP protocol & WB see attached file at bottom of enquiry

MATRIX USED protein A/G

DETERGENT NP40

AMOUNT OF PROTEIN LOADED 200ug - then sample loaded onto 2 blots - ~100ug for each blot

ELECTROPHORESIS/GEL CONDITIONS Reducing NuPAGE 4-12% gel

SECONDARY ANTIBODY [a competitor] AP conjugate both antimouse for monoclonal phospho antibodies and anti-rabbit for HDAC-2 (ab7029). 1/1000 dilution in 5%BSA-TBS-Tween(0.1%) for 1 hour at room temperature then wash 3x7 min in TBS-Tween(0.1%) + 1 x 5min in TBS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? none

ADDITIONAL NOTES please see blots & IP information Molecular weight marker is: Full range rainbow marker from Amersham.

ANSWER:

I'm sorry to hear you are experiencing problems with ab7029. Thank you for taking the time to fill in our online questionnaire with so many details and adding the image, this helped me a lot to understand the problem.

I am in touch with the laboratory to get more information about the protocol used when immunoprecipitating with ab7029 but have already been able to find out the following positive controls were tested: WB: nuclear extract of HeLa human epithelioid carcinoma cell line or whole extract of PC-12 rat pheochromocytoma cell line. Immunoprecipitation: whole lysate of NIH 3T cells.

The fact that you get several bands with the phospho-antibodies following immunoprecipitation is not surprising as a number of proteins tend to be co-immunoprecipitated with proteins which are involved in signalling cascades, such as HDAC2; and those co-IPed proteins tend to be also phosphorylated heavily. I think the main point we need to determine is whether the bands (1 and 3) you see when reprobing for total HDAC2 are correct.

Can you please clarify if the second blot "native HDAC2" (on the right) is a reprobe of the blot on the left, i.e. the same membrane? Have you tested the antibody ab7029 on immunoprecipitated HDAC2 directly or always after probing first for phospho-proteins? I would appreciate if you could clarify as if you have always re-probed the less- strong bands 4 and 5 could be due to "left over" staining with ab6626 and ab2286.

I think running the positive control of whole cell lysate of NIH 3T cells will help you determine if levels of HDAC2 are high in your samples, degraded or cleaved. You may need to consider a longer immunoprecipitation cycle, for example overnight incubation of the antibody with the lysate, so that you concentrate the protein to the maximum. The dilution 1/2500 is a recommendation with our own positive controls and you may need to try 1:1000.

I am asking the laboratory if they have further tips and apologise for the delay, I hope the above suggestions will already help,

I looked at the papers listed below the References for HDAC2 antibody (ab7029), it seems that none of them has been used in ChIP assay.

And you suggested me to use 1-8ug per 25ug chromatin but I don't know the concentration of this antibody. I didn't find any information about the concentration in the website or on the vial of the antibody. Could you please let me know the concentration of the antibody so I can decide where to start?

ANSWER:

Thank you for getting back to me.

Unfortunately we do not have details of the concentration of this serum. However, given that it is rabbit polyclonal serum you can assume that it is in the range of 10-16mgml-1.

I do know from past experience with this antiserum that it has an excellent titre. I look forward to hearing from you with regards your ChIP research. Good luck!

Please clarify the recommended dilution for ChIP - the ds states to use 2µl for 10 cells??

ANSWER:

Thank you for your enquiry.

It has been brought to my attention that ab7029, raised against HDAC2 has not yet been validated using Chromatin Immunoprecipitation (ChIP). I have therefore removed its status as "ChIP grade". That is not to say that it doesn’t work but that it is yet to be tested.

With regards your question as to a sensible starting dilution for your ChIP experiments, 1-8 µg of antiserum per 25 µg of chromatin often works well. I will be very interested in finding out how you get on in your ChIP experiments. Please can I encourage you to leave a review of how the antiserum performs by navigating to the product datasheet at Abcam.com and clicking on the reviews tab before following the instructions. In return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 points will be offered for an image).

If in time you have have difficulties with this antibody by ChIP please let me know and I will arrange for a credit note to be raised. I apologise for any inconvenience that this may have caused.

I look forward to hearing from you. Good luck with your research.

What is the typical IgG conentration of this product and ab7028 and ab7030. Also, when you specify that it can be used for immunoprecipitation, does this mean that it can be used in conjunction with assays to measure HDAC activity (i.e., that antibody binding does not interfere with enzyme activity)?

ANSWER:

I am afraid that we do not have the Ig concentrations for these antibodies. None of the antibodues have been tested in the way you describe, I believe they have just been tested with straight IP.