Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: MSH6 knockout  HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab208940 observed at 165 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab208940 was shown to specifically react with MSH6 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when MSH6 knockout samples were used. Wild-type and MSH6 knockout samples were subjected to SDS-PAGE.  Ab208940 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 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.

ab208940 staining MSH6 in wild-type HAP1 cells (top panel) and MSH6 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized 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 with ab208940 at 1μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling MSH6 with ab208940 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

Nuclear staining on tumor cells of human endometrium is observed [PMID: 8942985].

Counter stained with Hematoxylin.

Secondary antibody 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MSH6 with ab208940 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

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

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

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling MSH6 with ab208940 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

Nuclear staining on tumor cells of human colon cancer is observed [PMID: 26315971].

Counter stained with Hematoxylin.

Secondary antibody 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.

Anti-MSH6 antibody [EPR20316] (ab208940) at 1/1000 dilution + Human testis lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 153 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-MSH6 antibody [EPR20316] (ab208940) at 1/1000 dilution

Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 153 kDa
Observed band size: 120,160 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1,2 and 3: 3 minutes; Lane 4: 15 seconds.

This antibody recognizes isoforms 1 and 4 of MSH6. The observed molecular weight corresponds to isoform 1 (160kDa) and isoform 4 (120kDa) which is consistent with the Uniprot annotation.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling MSH6 with ab208940 at 1/500 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

MSH6 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab208940 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab208940 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: HeLa whole cell lysate, 10 µg (Input).

Lane 2: ab208940 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab208940 in HeLa whole cell lysate.

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

Exposure time: 8 seconds.

The observed molecular weight corresponds to isoform 1 (160kDa) and isoform 4 (120kDa) which is consistent with the Uniprot annotation.