Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling KAT3B / p300 with ab259330 at 1/500 (0.926 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing mainly nuclear staining in HeLa cells and no staining in HCT-15 cells. Negative control : HCT-15 (PMID : 14732695) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling KAT3B / p300 with ab259330 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling KAT3B / p300 with ab259330 at 1/500 (0.926 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling KAT3B / p300 with ab259330 at 1/500 dilution (0.1ug)/ red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.

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

Fresh lysates were used in this WB.

The ~100KDa signal is a non-target band.

Lane 2 of the blot was developed using a higher-sensitivity ECL substrate.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23753-55-ab259330'>ab259330</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Lane 2:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 10 µg

Lane 3:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 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

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.

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

Fresh lysates were used in this WB.

The ~100KDa signal is a non-target band.

Negative control : HCT15 (PMID : 14732695)

Exposure time : 3 minutes

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23753-55-ab259330'>ab259330</a>) at 1/1000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 2:

HCT15 (human colon epithelial cell), 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

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5 µg of ab259330 [EPR23753-55]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] - BSA and Azide free (AB283721)

This data was developed using ab259330, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : This antibody has no cross-reaction with human CREBBP.

Both recombinant proteins were made in-house.

The sample loaded onto lane 1 was purified EP300 recombinant protein expressed from an E.coli expression system.

The sample loaded onto lane 2 is E.coli extracts containing recombinant CREBBP protein.

15 seconds

Exposure time :

All lanes:

Western blot - Anti-KAT3B / p300 antibody [EPR23753-55] (<a href='/en-us/products/primary-antibodies/kat3b-p300-antibody-epr23753-55-ab259330'>ab259330</a>) at 1/1000 dilution

Lane 1:

His-tagged human EP300 recombinant protein at 0.01 µg

Lane 2:

His-tagged human CREBBP recombinant protein at 0.01 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

false