All lanes : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : TRIM24 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 116 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

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.

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.

All lanes : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TRIM24 knockout HeLa cell lysate
Lane 3 : Hap1 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 116 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

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 knockout cell line ab264963 (knockout cell lysate ab258246) lane below 140kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and TRIM24 knockout 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 : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution

Lane 1 : WEHI-3 (mouse leukemia lymphoblast), whole cell lysate
Lane 2 : P815 (mouse mastocytoma mast Cell), whole cell lysate
Lane 3 : CTLL-2 (mouse T lymphocyte), whole cell lysate
Lane 4 : C2C12 (mouse myoblasts myoblast), whole cell lysate
Lane 5 : Mouse colon tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 116 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

The expression profile observed is consistent with what has been described in the literature (PMID: 19909775). Blocking/Diluting buffer and concentration: 5% NFDM/TBST. Exposure Time: Lane 1: 3 minutes. Lanes 2-5: 15 seconds.

All lanes : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution

Lane 1 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 116 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

The expression profile observed is consistent with what has been described in the literature (PMID: 19909775). Blocking/Diluting buffer and concentration: 5% NFDM/TBST.  Exposure Time: 48 seconds.

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.

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.

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.