KAT3B/p300 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab275378 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275378 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 ug

Lane 2: ab275378 IP in K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab275378 in K-562 whole cell lysate

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

Exposure time: 76 seconds.

Lysate was freshly prepared and used in IP immediately to mininize protein degradation.

Incubation time was 2 hours.

Chromatin was prepared from K-562 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 ab275378 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labelling KAT3B/p300 with ab275378 at 1/100 (4.77 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000  dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling KAT3B/p300 with ab275378 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730)  isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) at 1/1000 dilution

Lane 1 : His-tagged human KAT3B/p300 recombinant protein (aa 1287-1666) 20 ng
Lane 2 : His-tagged human CREBBP recombinant protein, 10 µl

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 264 kDa

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

Both recombinant proteins were expressed from a E.coil expression system. Sample loaded onto lane 2 was E.coil extract containing CREBBP recombinant protein.

This product has weak cross reaction with CREBBP protein.

Exposure time: 5.5 seconds.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labelling KAT3B/p300 with ab275378 at 1/100 (4.77 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear and weakly cytoplasmic staining in HeLa cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

All lanes : Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) at 1/1000 dilution

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 264 kDa
Observed band size: 300 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 8995708).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure times: Lane 1: 125 seconds;Lanes 2-3: 3 minutes.