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)
Lane 2 : NIH 3T3 (Mouse) Whole Cell Lysate (ab52956)
Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957)
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate (ab28419) Then the membrane was incubated with alkaline phosphatase
Lane 5 : NIH 3T3 (Mouse) Whole Cell Lysate (ab52956) Then the membrane was incubated with alkaline phosphatase
Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) Then the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

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?


Exposure time: 8 minutes

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

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate. Then the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

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?

Blocking/Diluting Buffer and concentration

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).

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.

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

Lysates/proteins at 10 µg per lane.

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?

Blocking/Diluting Buffer and concentration 5% NFDM/TBST

HeLa (human epithelial cell line from cervix adenocarcinoma) or MCF7 (human breast adenocarcinoma cell line) cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/1000 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-mouse Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for

All lanes : 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
Lane 2 : HEK293T transfected with Tat containing plasmid whole cell lysate

Lysates/proteins at 30 µg per lane.

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 why is the actual band size different from the predicted?


Exposure time: 30 seconds

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.

See Abreview

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 .

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 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.