Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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 weak 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.

• ChIP

Supplier Data

ChIP - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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 weak 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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

Intracellular 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.

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using the same antibody clone in a different buffer formulation (ab275378 ).

Western blot : Anti-EP300 antibody [EPR23495-268] (ab275378) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab275378 was shown to bind specifically to EP300. A band was observed at 200/90-130 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EP300 knockout cell line. To generate this image, wild-type and EP300 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23495-268-chip-grade-ab275378'>ab275378</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EP300 knockout HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human EP300 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ep300-knockout-hct116-cell-line-ab286395'>ab286395</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Human brain olfactory lobe cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 200 kDa,90-130 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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.

All lanes:

Immunoprecipitation - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23495-268-chip-grade-ab275378'>ab275378</a>)

Predicted band size: 264 kDa

Observed band size: 300 kDa

false

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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

Both recombinant proteins were expressed from a E.coil expression system. Sample loaded onto lane2 was E.coil extract containing CREBBP recombinant protein.

This product has weak cross reaction with CREBBP protein.

Exposure time : 5.5 seconds.

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23495-268-chip-grade-ab275378'>ab275378</a>) at 1/1000 dilution

Lane 1:

His-tagged human KAT3B/p300 recombinant protein 20 ng

Lane 2:

His-tagged human CREBBP recombinant protein, 20 ng

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 264 kDa

Observed band size: 88.7 kDa,46.3 kDa

false

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade - BSA and Azide free (AB275388)

This data was developed using ab275378, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23495-268-chip-grade-ab275378'>ab275378</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 264 kDa

Observed band size: 300 kDa

false