Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling MSH2 with ab227941 at 1/8000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in para-carcinoma colonic epithelium (image B) or stromal cells (both image A and B) and loss of expression in the paired colon cancer (image A) (PMID: 24710284). Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-MSH2 antibody [EPR21017-123] (ab227941) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human tonsil lysate
Lane 5 : A-375 (human malignant melanoma epithelial cell), whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 104 kDa
Observed band size: 104 kDa

Exposure times: Lane 1: 100 seconds; Lane 2: 3 minutes; Lane 3: 10 seconds; Lane 4: 1 minute; Lane 5: 15 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-MSH2 knockout cells (red line) stained with ab227941. The cells were fixed with 80% methanol (5 min) (left panel) or 4% formaldehyde (10 min) (right panel) , and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by ab227941 for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MSH2  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

Note: We recommend fixing cells using MeOH instead of PFA to get optimal results.

Immunofluorescent analysis of 100% methanol-fixed A549 (human lung carcinoma cell line) cells labeling MSH2 with ab227941 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Nuclear staining in A549 cell line is shown.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling MSH2 with ab227941 at 1/8000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human testis was observed (PMID: 10029069). Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

MSH2 was immunoprecipitated from 0.35 mg of A-375 (human malignant melanoma cell line) lysate with ab227941 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227941 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: A-375 whole cell lysate 10 µg (Input). 

Lane 2: ab227941 IP in A-375 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227941 in A-375 whole cell lysate.

Exposure time: 1 second.

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

Immunofluorescent analysis of 100% methanol-fixed A-375 (human malignant melanoma cell line) cells labeling MSH2 with ab227941 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Nuclear staining in A-375 cell line is shown.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A-375 (human malignant melanoma cell line) cell line labeling MSH2 with ab227941 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled 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.