Immunohistochemical staining of paraffin embedded human colon with purified ab92471 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MSH6 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 163 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab92471 observed at 160 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab92471 was shown to react with MSH6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255410 (knockout cell lysate ab263763) was used. Wild-type HeLa and MSH6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab92471 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : MSH6 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 163 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab92471 observed at 160 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab92471 was shown to specifically react with MSH6 in wild-type HAP1 cells. No band was observed when MSH6 knockout samples were used. Wild-type and MSH6 knockout samples were subjected to SDS-PAGE. ab92471 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

ab92471 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 ab92471 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 staining of paraffin embedded rat liver with purified ab92471 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab92471 staining MSH6 in HeLa cells. 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 ab92471 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).

Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution (purified) + Rat brain at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 163 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution

All lanes : HAP1 cell line

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : donkey anti-rabbit IgG-HRP at 1/3000 dilution

Developed using the ECL technique.

Predicted band size: 163 kDa


Exposure time: 5 minutes

See Abreview

purified at 1/6000 dilution + SW480 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 163 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Anti-MSH6 antibody [EPR3945] (ab92471) at 1/2000 dilution (unpurified) + SW480 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 163 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Unpurified ab92471, at a 1/100 dilution, detecting MSH6 in paraffin embedded Human colonic adenocarcinoma tissue by immunohistochemistry. Detection used HRP conjugated anti rabbit antibody.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Unpurified ab92471 (1/500) staining MSH6 in asynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see abreview.

See Abreview

All lanes : Anti-MSH6 antibody [EPR3945] (ab92471) at 1/1000 dilution (unpurified)

Lane 1 : A431 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : SW480 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit antibody at 1/2000 dilution

Predicted band size: 163 kDa

Secondary antibody - goat anti-rabbit HRP (ab6721)

Equilibrium disassociation constant (K_D)
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