Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - BSA and Azide free

7 product images

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  ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - BSA and Azide free (ab264109)

  This image was produced using the same antibody clone but in a different formulation, Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade ab5408.
  

  Chromatin was prepared from U2OS 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 Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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] - BSA and Azide free (ab264109)

  All lanes: Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (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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  Flow Cytometry - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - BSA and Azide free (ab264109)

  Flow cytometry overlay histogram showing HeLa cells stained with ab264109 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10 % normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab264109) (1x10⁶ in 100 µL at 0.2 µg/ml) for 30 min at 22°C.

  The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) was used at 1/2000 for 30 min at 22°C.

  Isotype control antibody (black line) was mouse IgG1κ; (Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control ab170190) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

  Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

  This antibody gave a positive signal in HeLa cells fixed with 4 % formaldehyde (10 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions./

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  ELISA - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - BSA and Azide free (ab264109)

  This image was produced using the same antibody clone but in a different formulation, Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade ab5408.
  

  ELISA using Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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] - BSA and Azide free (ab264109)

  This image was produced using the same antibody clone but in a different formulation, 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 Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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] - BSA and Azide free (ab264109)

  This image was produced using the same antibody clone but in a different formulation, Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade ab5408.
  

  Overlay histogram showing HeLa cells stained with Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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 (Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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] - BSA and Azide free (ab264109)

  This data was developed using the same antibody clone in a different buffer formulation containing BSA and sodium azide (Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade ab5408). Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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 Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade 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.