Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labeling TRIM24 with ab256491 at 1/100 5.4 μg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 2 μg/ml dilution (Green). Confocal image showing nuclear staining in HepG2 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - 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 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 2 μg/ml dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab256491).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Wild type HAP1 (Wild type human chronic myelogenous leukemia near-haploid cell line) and TRIM24 knockout HAP1 (TRIM24 knockout human chronic myelogenous leukemia near-haploid cell line) cells labeling TRIM24 with ab256491 at 1/100 5.4 μg/ml dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 2 μg/ml dilution (Green). Confocal image showing nuclear staining in Wild type HAP1 cell line, and no staining in TRIM24 knockout HAP1 cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - 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 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 2 μg/ml dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab256491).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling TRIM24 with ab256491 at 1/500 dilution (1.08μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in human breast cancer (PMID : 21164480) is observed. The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab256491).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling TRIM24 with ab256491 at 1/500 dilution (1.08μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining in human lung cancer (PMID : 22666376) is observed. The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab256491).

• WB

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Western blot - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

This data was developed using the same antibody clone in a different buffer formulation (ab256491).

Lanes 1- 4 : Merged signal (red and green). Green - ab256491 observed at 140 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab256491 was shown to react with Tripartite Motif Containing 24 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264963 (CRISPR/Cas9 edited cell lysate ab258246) lane below 140kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and TRIM24 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab256491 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TRIM24 antibody [EPR22825-2] (<a href='/en-us/products/primary-antibodies/trim24-antibody-epr22825-2-ab256491'>ab256491</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

TRIM24 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human TRIM24 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-trim24-knockout-hela-cell-lysate-ab258246'>ab258246</a>)

Lane 2:

Western blot - Human TRIM24 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-trim24-knockout-hela-cell-line-ab264963'>ab264963</a>)

Lane 3:

Hap1 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 116 kDa

Observed band size: 140 kDa

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• WB

Lab

Western blot - Anti-TRIM24 antibody [EPR22825-2] - BSA and Azide free (AB260000)

The expression profile observed is consistent with what has been described in the literature (PMID : 19909775).

ab256491 was shown to specifically react with TRIM24 in wild-type HAP1 cells as signal was lost in TRIM24 knockout cells. Wild-type and TRIM24 knockout samples were subjected to SDS-PAGE. ab256491 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique. Blocking/Diluting buffer and concentration : 5% NFDM/TBST. Exposure Time : 48 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab256491).

All lanes:

Western blot - Anti-TRIM24 antibody [EPR22825-2] (<a href='/en-us/products/primary-antibodies/trim24-antibody-epr22825-2-ab256491'>ab256491</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

TRIM24 knockout HAP1 whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 116 kDa

Observed band size: 140 kDa

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