Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade

9 product images

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  ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Chromatin was prepared from U-2 OS (human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab5408 (blue), and 20 μl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

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  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Blocking/Diluting Buffer and concentration

  All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

  Lane 1: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 10 µg

  Lane 2: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate. Then the membrane was incubated with alkaline phosphatase at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution

  Predicted band size: 217 kDa

  Observed band size: 270 kDa

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  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) at 10 µg

  Lane 2: NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) at 10 µg

  Lane 3: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 10 µg

  Lane 4: MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) Then the membrane was incubated with alkaline phosphatase at 10 µg

  Lane 5: NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) Then the membrane was incubated with alkaline phosphatase at 10 µg

  Lane 6: PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) Then the membrane was incubated with alkaline phosphatase at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution

  Predicted band size: 217 kDa

  Observed band size: 270 kDa

  Exposure time: 8min

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  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Blocking/Diluting Buffer and concentration 5% NFDM/TBST
  
  

  All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

  Lane 2: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate. Then the membrane was incubated with alkaline phosphatase at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-mouse IgG at 1/10000 dilution

  Predicted band size: 217 kDa

  Observed band size: 270 kDa

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  This image is courtesy of an anonymous customer review.

  Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  7.5% SDS gel.
  

  Blocked with 5% BSA for 1 hour at 25°C.

  Incubated with the primary antibody for 3 hours at 25°C in 1X TBST buffer.

  All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/800 dilution

  Lane 1: HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 30 µg

  Lane 2: HEK293T transfected with Tat containing plasmid whole cell lysate at 30 µg

  Secondary

  All lanes: HRP-conjugated goat anti-mouse IgG at 1/5000 dilution

  Performed under reducing conditions.

  Predicted band size: 217 kDa

  Observed band size: 260 kDa

  Exposure time: 30s

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  ELISA - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  ELISA using ab5408 at varying antibody concentrations.
  
  Curve_SPL4 indicates binding to RNA polymerase II CTD repeat YSPTSPS peptide - phospho S5 (Human RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide ab18488).
  
  Binding to the following peptides was much weaker: Curve_SPL5 RNA polymerase II CTD repeat YSPTSPS peptide - phospho S2 (S. cerevisiae RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide ab12793), Curve_SPL6 RNA polymerase II CTD repeat YSPTSPS peptide (ab12795).

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  Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Dot blot analysis of RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide (Lane 1), RNA polymerase II CTD repeat YSPTSPS (phospho 2) phospho peptide (Lane 2) and RNA polymerase II CTD repeat YSPTSPS non-phospho peptide (Lane 3) labeling RNA polymerase II CTD repeat YSPTSPS (phospho S5) phospho peptide with ab5408 at a dilution of 1/1000 dilution (1ug/ml). A HRP-conjugated goat anti-mouse IgG was used as the secondary antibody at a dilution of 1/10,000 dilution.
  

  Blocking buffer: 5% NFDM/TBST. Dilution buffer: 5% NFDM /TBST .

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  Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab5408 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5408, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  
  

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  Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408)

  ab5408 staining RNA polymerase II CTD repeat YSPTSPS (phospho S5) in HeLa cells. The cells were fixed with 100% methanol (5 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 ab5408 at 1µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min).Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.