Unpurified ab92312 at 1/100 dilution staining MLH1 in Human colonic adenocarcinoma by Immunohistochemistry, Paraffin-embedded tissue. The use of an HRP/AP polymerized antibody is recommended for a secondary antibody. Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

All lanes : Anti-MLH1 antibody [EPR3894] (ab92312) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MLH1 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 84 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab92312 observed at 90 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab92312 Anti-MLH1 antibody [EPR3894] was shown to specifically react with MLH1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab267223 (knockout cell lysate ab257172) was used. Wild-type and MLH1 knockout samples were subjected to SDS-PAGE. ab92312 and Anti-GAPDH antibody [6C5] - Loading Control were incubated overnight at 4°C at 1 in 10000 dilution and 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-MLH1 antibody [EPR3894] (ab92312) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : MLH1 knockout HAP1 cell lysate
Lane 3 : HCT116 cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 84 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab92312 observed at 88 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab92312 was shown to recognize MLH1 in wild-type HAP1 cells along with additonal cross reactive bands. No band was observed when MLH1 knockout samples were examined. Wild-type and MLH1 knockout samples were subjected to SDS-PAGE. ab92312 and ab8245 (loading control to GAPDH) were both diluted 1/1000 and incubated overnight at 4°C. 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human colon tissue sections labeling MLH1 with Purified ab92312 at 1:250 dilution (2.9 µg/ml). Heat mediated antigen retrieval was performed using citrate (pH 6.0)ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunocytochemistry/ Immunofluorescence analysis of SW480 (Human colorectal adenocarcinoma epithelial cell) cells labeling MLH1 with Purified ab92312 at 1:500 dilution (1.6 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor®594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor®488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-MLH1 antibody [EPR3894] (ab92312) at 0.4 µg/ml (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HCT116 (Human colorectal carcinoma epithelial cell) whole cell lysates, negative control
Lane 3 : Human testis lysates
Lane 4 : Rat testis lysates
Lane 5 : Mouse thymus lysates
Lane 6 : Rat thymus lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 84 kDa

Blocking and diluting buffer: 5% NFDM/TBST.
According to PMID:23653048, HCT116 is MLH1 negative cell line.

Overlay histogram showing HeLa cells stained with unpurified ab92312 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92312, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-MLH1 antibody [EPR3894] (ab92312) at 1/10000 dilution (Unpurified)

Lane 1 : 293 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : SW480 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 84 kDa

Unpurified ab92312 staining MLH1 in Human colorectal (top) and gastric tissue (bottom) by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Unpurified ab92312 at 1/100 dilution staining MLH1 in Human tonsil by Immunohistochemistry, Paraffin-embedded tissue. The use of an HRP/AP polymerized antibody is recommended for a secondary antibody. Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling MLH1 with purified ab92312 at 1/1000. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

Unpurified ab92312 (1/200) staining MLH1 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to abreview.

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