Anti-MST4 antibody [EP1864Y] (ab52491) at 1/200000 dilution + HeLa cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labelling MST4 with purified ab52491 at 1/500. Cells were fixed with 100% methanol and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

MST4 was immunoprecipitated using ab52491 at 1:100 dilution (2μg in 0.35mg lysates).

All lanes : Anti-MST4 antibody [EP1864Y] (ab52491) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with 5% NFDM/TBST
Lane 2 : HeLa whole cell lysate immunoprecipitated with ab52491 1:100 dilution (2µg in 0.35mg lysates) with 5% NFDM/TBST
Lane 3 : HeLa whole cell lysate immunoprecipitated with Rabbit monoclonal IgG (ab172730) 1:100 dilution (2µg in 0.35mg lysates), with 5% NFDM/TBST

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/5000 dilution (VeriBlot for IP secondary antibody (HRP))

Exposure time: 10 seconds

Overlay histogram showing HeLa cells stained with ab52491 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52491, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical analysis of paraffin-embedded human placenta using ab52491 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-MST4 antibody [EP1864Y] (ab52491) at 1/20000 dilution + Recombinant human MST4 protein (ab60341) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Predicted band size: 47 kDa


Exposure time: 20 seconds


Ab52491 recognizes the full length tagged recombinant MST4 protein (ab60341) which has an expected molecular weight of 72 kDa.

All lanes : Anti-MST4 antibody [EP1864Y] (ab52491) at 1/100000 dilution

Lane 1 : Mouse brain tissue lysate with 5% NFDM/TBST
Lane 2 : Rat brain tissue lysate with 5% NFDM/TBST

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds