Anti-MSH2 抗体 [EPR21017-123]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-MSH2 antibody [EPR21017-123] (AB227941)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized A-375 (humanmalignant 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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 MeOHinstead of PFA toget optimal results.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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.

• IP

Unknown

Immunoprecipitation - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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.

All lanes:

Immunoprecipitation - Anti-MSH2 antibody [EPR21017-123] (ab227941)

Predicted band size: 105 kDa

Observed band size: 105 kDa

true

• WB

Supplier Data

Western blot - Anti-MSH2 antibody [EPR21017-123] (AB227941)

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.

All lanes:

Western blot - Anti-MSH2 antibody [EPR21017-123] (ab227941) at 1/1000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

A549 (human lung carcinoma epithelial cell), whole cell lysate at 10 µg

Lane 3:

Human fetal heart lysate at 10 µg

Lane 4:

Human tonsil lysate at 10 µg

Lane 5:

A-375 (human malignant melanoma epithelial cell), whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 105 kDa

Observed band size: 104 kDa

true

• WB

Lab

Western blot - Anti-MSH2 antibody [EPR21017-123] (AB227941)

Western blot : Anti-MSH2 antibody [EPR21017-123] (ab227941) 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, ab227941 was shown to bind specifically to MSH2. A band was observed at 104 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MSH2 knockout cell line. To generate this image, wild-type and MSH2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-MSH2 antibody [EPR21017-123] (ab227941) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

MSH2 knockout HCT 116 cell lysate at 20 µg

Lane 3:

A-375 cell lysate at 20 µg

Observed band size: 104 kDa

false