Anti-RNA polymerase II CTD repeat YSPTSPS antibody [H14] - ChIP Grade (ab24759)

Dear technical team,

I wondered which ligand you suggest for immunoprecipitation using your antibody ab24759 (anti-RNA polymerase II CTD repeat YSPTSPS antibody

[H14] - ChIP Grade)?

As far as I understand is your H14 antibody of IgM isotype which is poorly or not at all recognized by protein A or G.

Thanks in advance,

ANSWER:

Thank you for contacting us. Although as you say the IgM is not bound well by protein A or G this antibody has been used previously with Protein G and Protein A/G beads successfully for immunoprecipitation for performing ChIP:

Protein G beads:

Tao Y et al. Treatment of breast cancer cells with DNA demethylating agents leads to a release of Pol II stalling at genes with DNA-hypermethylated regions upstream of TSS. Nucleic Acids Res : (2011).  PubMed: 21880597

reference to protocol used in this reference:

http://www.pnas.org/content/104/36/14366.full.pdf+html

Protein A/G beads: Welboren WJ et al. ChIP-Seq of ERalpha and RNA polymerase II defines genes differentially responding to ligands. EMBO J : (2009). PubMed: 19339991

reference to protocol used in this reference:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1852848/?tool=pubmed

There are alternatives available which can be used in the immunoprecipitation such as protein L or using an IgG anti-IgM with Protein A or G beads. Protein L is a protein which binds the light chains of immunoglobulins while Protein A and G bind the heavy chain. More information on these two alternative purification systems can be found from this link:

http://www.abcam.com/ps/pdf/protocols/igm_purification.pdf

I hope this information has been of help. If you have any further questions please do not hesitate to ask.

Are these antibodies tested in mouse, as the datasheet says tested in 'many'

ANSWER:

Thank you for your telephone enquiry yesterday.

I have investigated this further and can confirm that both these RNA POL II antibodies (ab24758 and ab24759) have been tested to detect mouse RNA POL II

I have included a reference below that uses some POLII antibodies, including these ones, with 3T3 cells.

The Oncogenic TLS-ERG Fusion Protein Exerts Different Effects in Hematopoietic Cells and Fibroblasts Junhui Zou, Hitoshi Ichikawa, Michael L. Blackburn, Hsien-Ming Hu, Anna Zielinska-Kwiatkowska, Qi Mei, Gerald J. Roth, Howard A. Chansky, and Liu Yang Mol. Cell. Biol., Jul 2005; 25: 6235 - 6246.

I hope this is helpful to you. Should you have any further questions, please do not hesitate to contact us.

I have a question about the orgin of the H5 and H14 antibodies (I didn't know which kind of comment I should choose) against the phosphorylated forms of Pol II CTD (ab24758 and ab24759). The original papesr from Bregman DB 1994 and Warren SL 1992 call the H5 and H14 epitopes cytostallin, and somehow in 1995 it became RNA polymerase II. Are these really the same antibodies which are sold by Abcam, and if so, when and how did people show that cytostallin is Pol II ?

ANSWER:

Thank you for your enquiry.

I apologise for the delay in responding to your message. It has taken some time to obtain some information.

I can confirm that when these H5 and H14 antibodies were first used, they detected a protein initially named cytostellin. Although it was shown to be a protein of 240kd (same as large subunit RNA pol II), it was believed to be a novel protein because it stained locations in the nucleus where RNA pol II was believe to be exluded. Subsequent protein sequencing cleared up the confusion, showing that what had been named cystostellin was in fact RNA pol II large subunit.

A history of these events is described in the Description section of the following website:

http://www.patentstorm.us/patents/6007985-description.html

I hope this information is helpful. Should you have any further questions, please do not hesitate to contact us again.

BATCH NUMBER 136696 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM There was no signal observed in western blot of total cell lysate, nuclear lysate or insoluble pellet of 4 different human cell lines U2OS, Hela, 293 and 293T. Also there was no detectable signal in immunofluorescence.

SAMPLE Human/cell and nuclear extract

PRIMARY ANTIBODY Abcam/mouse monoclonal/3%BSA TBST/1:500/ 1 hour and overnight/ 3 washes in TBST prior to 2? antibody addition.

DETECTION METHOD ECL- Pierce Dura kit

POSITIVE AND NEGATIVE CONTROLS USED All cell lines are far as I know should be positive for RNA pol II.

ANTIBODY STORAGE CONDITIONS Aliquot and store at -20?C

SAMPLE PREPARATION NP40 lysis buffer/protease cocktail inhibitors + Mg132 + PMSF/ sample heated in Laemli loading buffer to denature - 5minutes at 99?C.

AMOUNT OF PROTEIN LOADED 50ug of total cell lysate or nuclear lysate and whole insoluble pellet from 10^6 cells.

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 7%

TRANSFER AND BLOCKING CONDITIONS Transfer was complete as determined by prestained markers. The membrane was also after probed for tubulin, which was there although this is only a 55kDa protein. The blocking agent was 3% BSA in TBST.

SECONDARY ANTIBODY Dianova /HRPO goat anti mouse/1.5%BSA TBST/1:20000/ 1 hour/ 3 washes in TBST (10 min. each)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have used total cell lysate, nuclear lysate and insoluble fraction of cell collections as well as immunofluorescence and no signal was detected in any. I also tried the antibody in immunofluorescense in a 1:100 dilution. Molecular probes AlexaFluor 588 goat anti mouse secondary antibody in a 1:1000 dilution was used and no signal was observed.

ADDITIONAL NOTES I also tried the antibody in immunofluorescense in a 1:100 dilution. Molecular probes AlexaFluor 588 goat anti mouse secondary antibody in a 1:1000 dilution was used and no signal was observed. Furthermore I have attached one immunofluorescence pictures (one with long exposure time). Only after 13,000ms can the any red signal be seen. At 3,000ms no signal is observed (not shown). The signal that is observed after 13,000ms is not the correct localisation of RNA pol II and is only background. I have not attached a picture of the western blots as they were blank. There is only 100ul of the antibody provided and the prementioned experiments were time and cost expensive and so I will not perform anymore experiments with this antibody. I am requesting either a refund or a different functioning antibody lot.

ANSWER:

Thank you for your enquiry. I don't see any problems with your customer's protocol and we have not received reports of other complaints regarding this antibody. It seems that perhaps that the vial your customer received was damaged in some way.

I would be happy to send a free of charge replacement vial of issue you a credit if your customer prefers a refund. Please let me know how they would like to proceed.