Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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.

• ChIP

Supplier Data

ChIP - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

Chromatin was prepared from K-562 cells according to theAbcam 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).

• IP

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Immunoprecipitation - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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 (ab275378)

Predicted band size: 264 kDa

Observed band size: 300 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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 Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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 (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 (ab275378) 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

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• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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 (ab275378) 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

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• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

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.

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (ab275378) 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

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• WB

CiteAb

Western blot - Anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade (AB275378)

KAT3B / p300 western blot using anti-KAT3B / p300 antibody [EPR23495-268] - ChIP Grade ab275378. Publication image and figure legend from Sherman, E. J., Mirabelli, C., et al., 2022, PLoS Pathog, PubMed 35231079.

ab275378 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab275378 please see the product overview.

Regulation of ACE2 by SMAD4, EP300, PIAS1, and BAMBI is mediated at the mRNA level.(A) Immunoblot of lysates collected from HuH7 cells targeted by CRISPR with a gRNA against the indicated gene or a nontargeting (NT) control sequence. (B) Proportion of saponin-permeabilized cells exhibiting ACE2 staining above background after CRISPR targeting of the indicated gene in ACE2-enriched HuH7 cells. (C) Immunofluorescence microscopy of ACE2 staining (green) in ACE2-enriched HuH7 cells either untreated (WT) or targeted by CRISPR with a gRNA against the indicated gene or a nontargeting (NT) control sequence. Scale bar = 100 μm. (D) Quantification of relative ACE2 mRNA levels in the indicated cell lines by qRT-PCR of ACE2 mRNA, normalized to the mean Ct for a panel of control transcripts (RPL37, RPL38, and ACTB). Asterisks indicate p<0.05 relative to nontargeting controls by Student's t-test. Individual data points in panels B and D represent technical replicates.

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