Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336]

• ICC/IF

AbReview12245****

Immunocytochemistry/ Immunofluorescence - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

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.

This image is courtesy of an anonymous Abreview

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

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 >5000 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.

• IP

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Immunoprecipitation - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

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.

All lanes:

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

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

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).

• WB

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Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/2000 dilution

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

Jurkat whole cell lysate at 20 µg

Lane 3:

293T whole cell lysate at 20 µg

Lane 4:

MCF-7 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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• WB

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Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

All lanes:

Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/5000 dilution

All lanes:

293T cell lysate.

Observed band size: 110 kDa

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• WB

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Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

Blocking and Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (ab32519) at 1/2000 dilution

Lane 1:

NIH/3T3 whole cell lysate at 20 µg

Lane 2:

C6 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa

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• WB

Supplier Data

Western blot - Anti-Myosin Phosphatase + Myosin Phosphatase 2 antibody [YE336] (AB32519)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - 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 at 0.01 µg

Lane 2:

Human Myosin Phosphatase 2 full-length recombinant protein with DDDDK tag at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 150 kDa

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Exposure time: 5s