RNA polymerase II CTD repeat YSPTSPS was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab252854 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252854 at 1/1000 dilution. Goat Anti-Rat IgG (H+L), HRP) (ab205720) was used at 1/5000 dilution.

Lane 1: Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 ug

Lane 2: ab252854 IP in Raji whole cell lysate

Lane 3: Rat monoclonal IgG2a (ab18450) instead of ab252854 in Raji whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 24 seconds

 

Anti-RNA polymerase II CTD repeat YSPTSPS antibody [1C7] (ab252854) at 1/1000 dilution + PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Secondary
Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution

Predicted band size: 217 kDa
Observed band size: 250 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 22745433).

Exposure time: 3 minutes

Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab252854 (red), or 5 µg of Rat IgG2a ab18450 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

RNA polymerase II CTD repeat YSPTSPS was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab252854 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252854 at 1/1000 dilution. Goat Anti-Rat IgG (H+L), HRP) (ab205720) was used at 1/5000 dilution.

Lane 1: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 ug

Lane 2: ab252854 IP in RAW264.7 whole cell lysate

Lane 3: Rat monoclonal IgG2a (ab18450) instead of ab252854 in RAW264.7 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 24 seconds

 

All lanes : Anti-RNA polymerase II CTD repeat YSPTSPS antibody [1C7] (ab252854) at 1/1000 dilution

Lane 1 : Raji (human Burkitts lymphoma B lymphocyte), whole cell lysate
Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG (H+L), HRP) (ab205720) at 1/5000 dilution

Predicted band size: 217 kDa
Observed band size: 250 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST

The expression profile/ molecular weight  observed is consistent with what has been described in the literature (PMID: 22745433)

This blot was developed using a higher sensitivity ECL substrate. 

Exposure time: 37 seconds

ELISA analysis using ab252854 at a range of 4000-0 ng/ml followed by a Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rat IgG (H+L) at 1/1000 dilution.

Antigens: phospho S2, phospho S5, phospho T4, phospho Y1, phospho S7, non-phospho

Dot blot analysis of ab252854 at 1/1000 dilution followed by a Goat Anti-Rat IgG (H+L), HRP) (ab205720) Secondary antibody at 1/5000 diluton. The observed data is consistent with what has been described in the literature (PMID: 22549466). Exposure trime: 3 minutes.

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1: RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide
Lane 2: RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide
Lane 3: RNA polymerase II CTD repeat YSPTSPS (phospho T4) peptide
Lane 4: RNA polymerase II CTD repeat YSPTSPS (phospho Y1) peptide
Lane 5: RNA polymerase II CTD repeat YSPTSPS (phospho S7) peptide
Lane 6: RNA polymerase II CTD repeat YSPTSPS non-phopho peptide