Anti-MLH1 antibody [EPR3894] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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] - BSA and Azide free (AB214441)

Unpurified ab92312 staining MLH1 in human colorectal (top) and gastric tissue (bottom) by immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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

• WB

Supplier Data

Western blot - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/mlh1-antibody-epr3894-ab92312'>ab92312</a>) 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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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. Hematoxylin was used as a counterstain

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• ICC/IF

AbReview29256****

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92312).

• WB

Lab

Western blot - Anti-MLH1 antibody [EPR3894] - BSA and Azide free (AB214441)

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] (<a href='/en-us/products/primary-antibodies/mlh1-antibody-epr3894-ab92312'>ab92312</a>) 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

false