Recombinant Anti-MLH1 antibody [EPR3894] (ab92312)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and MLH1 knockout Hap1 stained with ab92312 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab92312) (1x 10⁶ in 100μl at 0.2 μg/ml (1/9835)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, MLH1 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

False colour image of Western blot: Anti-MLH1 antibody [EPR3894] staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab92312 was shown to bind specifically to MLH1. A band was observed at 85 kDa in wild-type A549 cell lysates with no signal observed at this size in MLH1 CRISPR-Cas9 edited cell line ab276105 (CRISPR-Cas9 edited cell lysate ab283566). The band observed in the CRISPR-Cas9 edited lysate lane below 85 kDa is likely to represent a truncated form of MLH1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MLH1 CRISPR-Cas9 edited A549 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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.

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.

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

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

Overlay histogram showing HeLa cells stained with unpurifiedab92312 (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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. 

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.

See Abreview

Tissue Microarrays stained for "Anti-MLH1 antibody [EPR3894]” using "ab92312" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0). The sections were incubated with ab92312 at +4°C overnight. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.