Anti-MLH1 antibody [EPR3894]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - 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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] (AB92312)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] (AB92312)

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

Image from Wang X et al. PLoS One. 2011;6(10):e25913. Epub 2011 Oct 12. Fig 3.; doi:10.1371/journal.pone.0025913; October 12 2011 PLoS ONE 6(10): e25913.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

• ICC/IF

AbReview29256****

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

This image is courtesy of an Abreview submitted by Kirk McManus.

• WB

Supplier Data

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

All lanes:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

MLH1 knockout HAP1 cell lysate at 20 µg

Lane 3:

HCT116 cell lysate at 20 µg

Lane 4:

293T cell lysate at 20 µg

Predicted band size: 85 kDa

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• WB

Lab

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

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.

All lanes:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MLH1 CRISPR-Cas9 edited A549 cell lysate at 20 µg

Lane 2:

Western blot - Human MLH1 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-mlh1-knockout-a549-cell-line-ab276105'>ab276105</a>)

Lane 2:

Western blot - Human MLH1 knockout A549 cell lysate (<a href='/en-us/products/cell-lysates/human-mlh1-knockout-a549-cell-lysate-ab288239'>ab288239</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HCT 116 cell lysate at 20 µg

Predicted band size: 85 kDa

Observed band size: 85 kDa

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• WB

Lab

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HeLa whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab92312 (1/10,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab92312 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 1/10000 dilution

All lanes:

HeLa whole cell lysate at 15 µg with NDFM/TBST

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 1/20000 dilution

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Exposure time: 20s

• WB

Supplier Data

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

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:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MLH1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MLH1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mlh1-knockout-hela-cell-line-ab267223'>ab267223</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HCT116 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 85 kDa

false

• WB

Unknown

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

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

All lanes:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 0.4 µg/mL

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HCT116 (Human colorectal carcinoma epithelial cell) whole cell lysates, negative control at 20 µg

Lane 3:

Human testis lysates at 20 µg

Lane 4:

Rat testis lysates at 20 µg

Lane 5:

Mouse thymus lysates at 20 µg

Lane 6:

Rat thymus lysates 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 1/20000 dilution

Predicted band size: 85 kDa

false

• WB

Unknown

Western blot - Anti-MLH1 antibody [EPR3894] (AB92312)

All lanes:

Western blot - Anti-MLH1 antibody [EPR3894] (ab92312) at 1/10000 dilution

Lane 1:

293 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

A431 cell lysate at 10 µg

Lane 4:

SW480 cell lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 85 kDa

false