Anti-MLH1 antibody [EPR20522]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MLH1 antibody [EPR20522] (AB223844)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and MLH1 knockout Hap1 stained with ab223844 (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 (ab223844) (1x 106 in 100μl at 1.0 μg/ml (1/2070)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR20522] (AB223844)

Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue labelling MLH1 with ab223844 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in tumor cells of human ovarian cancer (PMID : 10778972) is observed. Counter stained with hematoxylin.

Secondary antobody only control : used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MLH1 antibody [EPR20522] (AB223844)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HCT 116 (human colorectal carcinoma epithelial cell line, left) / SW480 (human colorectal adenocarcinoma cell line, right) cells labelling MLH1 with ab223844 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077)at 1/2000 dilution was used as the secondary antibody.

The HCT 116 (human colorectal carcinoma epithelial) cell line is a negative control for MLH1 (PMID : 23724141) (left panel).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR20522] (AB223844)

Immunohistochemical analysis of paraffin-embedded human colon tissue labelling MLH1 with ab223844 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in human colon is observed (PMID : 10535979). Counter stained with hematoxylin.

Secondary antobody only control : used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR20522] (AB223844)

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labelling MLH1 with ab223844 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in tumor cells of human colon cancer is observed (PMID : 22608206). Counter stained with hematoxylin.

Secondary antobody only control : used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MLH1 antibody [EPR20522] (AB223844)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelling MLH1 with ab223844 at 1/100 (7.79 μg/ml) dilution for 10 minutes at room temperature, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on human breast carcinoma is observed. Counter stained with hematoxylin.The immunostaining was performed on a Leica Biosystems BOND® RX instrument Secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Perform heat mediated antigen retrieval with Tris/EDTA buffer (pH 9.0,Epitope Retrieval Solution2) for 10 minutes before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR20522] (AB223844)

Immunofluorecent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilised SW480 (human colorectal adenocarcinoma cell line) cells labelling MLH1 with ab223844 at 1/100 dilution followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution (green). Confocal image showing nuclear staining in SW480 cell line.

DAPI was used as the Nuclear counterstain (blue). Alpha Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

The HCT 116 (human colorectal carcinoma epithelial) cell line is a negative control for MLH1 (PMID : 23724141).

Secondary antibody only control : PBS instead of ab223844 followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MLH1 antibody [EPR20522] (AB223844)

ab223844 staining MLH1 in wild-type Hap1 cells, with negative expression in MLH1 knockout Hap1 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab223844 at 0.2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IP

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Immunoprecipitation - Anti-MLH1 antibody [EPR20522] (AB223844)

MLH1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab223844 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223844 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (input).
Lane 2 : ab223844 IP in HeLa whole cell lysate.
Lane 3 : Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab223844 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : Less than 1 second.

All lanes:

Immunoprecipitation - Anti-MLH1 antibody [EPR20522] (ab223844)

Predicted band size: 85 kDa

Observed band size: 84 kDa

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• WB

Lab

Western blot - Anti-MLH1 antibody [EPR20522] (AB223844)

Lanes 1 - 4 : Merged signal (red and green). Green - ab223844 observed at 85 kDa. Red - loading control, ab9484, observed at 37 kDa

ab223844 was shown to specifically react with MLH1 in wild-type HAP1 cells as signal was lost in MLH1 knockout cells. Wild-type and MLH1 knockout samples were subjected to SDS-PAGE. ab223844 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MLH1 antibody [EPR20522] (ab223844) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

MLH1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HCT116 whole cell lysate at 20 µg

Lane 4:

Hek293 whole cell lysate at 20 µg

Predicted band size: 85 kDa

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• WB

Supplier Data

Western blot - Anti-MLH1 antibody [EPR20522] (AB223844)

Exposure times.

Lanes 1-3 : 8 seconds
Lane 4 : 3 minutes.

The expression profile observed is consistent with the literature (PMID : 15249596). The HCT116 cell line is a negative control for MLH1 (PMID : 23724141).

All lanes:

Western blot - Anti-MLH1 antibody [EPR20522] (ab223844) at 1/1000 dilution

Lane 1:

SW480 (human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell line) whole cell lysate at 20 µg

Lane 3:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Human colon tissue lysate 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 1/100000 dilution

Predicted band size: 85 kDa

Observed band size: 84 kDa

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• WB

Lab

Western blot - Anti-MLH1 antibody [EPR20522] (AB223844)

False colour image of Western blot : Anti-MLH1 antibody [EPR20522] staining at 1/1000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot ab223844 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 ab288239). 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°. 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 [EPR20522] (ab223844) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

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>) at 20 µg

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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