All lanes : Anti-MSH6 antibody [44] (ab14204) at 1/500 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.

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

Lanes 1- 2: Merged signal (red and green). Green - ab14204 observed at 160 kDa. Red - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) observed at 50 kDa.

 ab14204 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. ab14204 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab14204 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 ab14204 at 1/250 dilution and ab202272 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 Mouse IgG (Alexa Fluor® 488) (ab150117) 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).

All lanes : Anti-AMACR + p63 antibody [4A4 (p63)] (ab14202)

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.

Lanes 1 - 4: Merged signal (red and green). Green - ab14204 observed at 160 kDa. Red - loading control, ab18251, observed at 52 kDa.

ab14204 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 HAP1 samples were used. Wild-type and MSH6 knockout samples were subjected to SDS-PAGE. ab14204 and ab18251 (loading control to alpha Tubulin) were diluted at 1/100 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab14204 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 ab14204 at 1/250dilution and ab202272 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 Mouse IgG (Alexa Fluor® 488) (ab150117) 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).

ab14204 staining human colon carcinoma by IHC-P.