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.

All lanes : Anti-MLH1 antibody [EPR20522] (ab223844) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : MLH1 knockout HAP1 whole cell lysate
Lane 3 : HCT116 whole cell lysate
Lane 4 : Hek293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 84 kDa

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

Lane 1 : SW480 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 2 : HCT 116 (human colorectal carcinoma epithelial cell line) whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Human colon tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 84 kDa
Observed band size: 84 kDa

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).

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.

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.

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.

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).

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.