Anti-Myosin Phosphatase + Myosin Phosphatase 2 抗体 [YE336] (ab32519)

All lanes : Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/1000 dilution

Lane 1 : Human Myosin Phosphatase 1 full-length recombinant protein with DDDDK tag
Lane 2 : Human Myosin Phosphatase 2 full-length recombinant protein with DDDDK tag

Lysates/proteins at 0.01 µg per lane.

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

Predicted band size: 110 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/2000 dilution

Lane 1 : HeLa whole cell lysate
Lane 2 : Jurkat whole cell lysate
Lane 3 : 293T whole cell lysate
Lane 4 : MCF-7 whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 110 kDa
Observed band size: 110 kDa



Blocking and Diluting buffer 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 cells labelling Myosin Phosphatase 1+Myosin Phosphatase 2 with purified ab32519 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291 anti-Tubulin (mouse mAb) followed by ab150120, AlexaFluor^®594 goat anti-mouse secondary both at 1/1000. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120). For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

All lanes : Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/2000 dilution

Lane 1 : NIH/3T3 whole cell lysate
Lane 2 : C6 whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 110 kDa
Observed band size: 110 kDa



Blocking and Diluting buffer 5% NFDM/TBST

Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/5000 dilution (unpurified) + 293T cell lysate.

Predicted band size: 110 kDa
Observed band size: 110 kDa

Intracellular Flow Cytometry analysis of HeLa cells labelling Myosin Phosphatase 1+Myosin Phosphatase 2 (red) with purified ab32519 at dilution of 1/40. The secondary antibody used was goat anti rabbit IgG (FITC) at 1/500. Cells were fixed with 4% paraformaldehyde. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody. 

 

 

ab32519 at 1/30 dilution immunoprecipitating Myosin Phosphatase 1+Myosin Phosphatase 2 in HeLa whole cell lysate observed at 110 KDa (lanes 1 and 2).

Lane 1 (input): HeLa whole cell lysate 10ug

Lane 2 (+): ab32519 + HeLa whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32519 in HeLa whole cell lysate

For western blotting, ab32519 was used followed by VeriBlot for IP Detection Reagent (HRP) (ab131366) for detection at a dilution of 1/10,000.

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

ab32519 staining mouse fibroblast cells by ICC/IF. Cells were PFA fixed, permeabilized in Triton X-100 and blocked in 1% BSA for 30 minutes at 25°C. The primary antibody was diluted 1/200 and incubated with sample for 4 hours at 25°C. An Alexa Fluor® 488 conjugated goat monoclonal to rabbit, diluted 1/500 was used as the secondary.

See Abreview

Overlay histogram showing HEK293 cells stained with unpurified ab32519 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab32519, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.