Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade

• ChIP

Unknown

ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 5μg of ab26721 (blue), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the inactive AFM and F8 promoters, the GAPDH promoter (active) and over the g-Actin gene (active). Schematic diagram of the g-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

IHC image of RNA polymerase II CTD repeat YSPTSPS staining in a section of formalin-fixed paraffin-embedded normal human Hodgkin's lymphoma* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab26721, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.*

Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• ICC/IF

AbReview24031****

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

ab26721 (1/200) staining RNA polymerase II in assyncchonous HeLa cells (green). cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

Image courtesy of an AbReview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• ICC/IF

AbReview51776****

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

ab26721 staining RNA polymerase II CTD in the human HeLa cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Tween-20 and blocked with 1% BSA for 10 minutes at 25°C. Samples were incubated with primary antibody (1/400 in TBS) for 12 hours at 4°C. A FITC conjugated anti-rabbit goat IgG polyclonal (1/500) was used as the secondary antibody.

This image is courtesy of an anonymous AbReview.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

ab26721 staining RNA polymerase II CTD repeat YSPTSPS in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab26721 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IP

Unknown

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

RNA polymerase II CTD repeat YSPTSPS was immunoprecipitated using 0.5mg Hela whole cell extract, 5μg of Rabbit polyclonal to RNA polymerase II CTD repeat YSPTSPS and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70^oC; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab26721.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to mouse anti-Rabbit HRP (IgG light chain) (ab99697).
Band : 220kDa; RNA polymerase II CTD repeat YSPTSPS.

All lanes:

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

Predicted band size: 217 kDa

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• WB

Ap

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (AB26721)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS peptide (<a href='/en-us/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-peptide-ab17564'>ab17564</a>)

Lane 4:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS peptide (<a href='/en-us/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-peptide-ab17564'>ab17564</a>)

Lane 5:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (<a href='/en-us/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s5-peptide-ab18488'>ab18488</a>)

Lane 6:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg with Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide (<a href='/en-us/products/proteins-peptides/human-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s5-peptide-ab18488'>ab18488</a>)

Lane 7:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (<a href='/en-us/products/proteins-peptides/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s2-peptide-ab12793'>ab12793</a>)

Lane 8:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 20 µg with S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide (<a href='/en-us/products/proteins-peptides/s-cerevisiae-rna-polymerase-ii-ctd-repeat-ysptsps-phospho-s2-peptide-ab12793'>ab12793</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 217 kDa

Observed band size: 220 kDa

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Exposure time: 1min