All lanes : 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 mg/ml
Lane 2 : NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) at 10 µg
Lane 3 : C12 (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 why is the actual band size different from the predicted?

This image was produced using the same antibody clone but in a different formulation, 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 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.

This image was produced using the same antibody clone but in a different formulation, ab5408.

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

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) (ab150117) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody (black line) was mouse IgG1κ; (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./

This image was produced using the same antibody clone but in a different formulation, 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 .

This image was produced using the same antibody clone but in a different formulation, ab5408.

Overlay histogram showing HeLa 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 22ºC. The secondary antibody used was goat anti-mouse DyLight® 488 (IgG H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.