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Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human Myosin Phosphatase 1+Myosin Phosphatase 2 (C terminal).
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
Our Abpromise guarantee covers the use of ab32519 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/2000. Predicted molecular weight: 110 kDa.
For unpurified use at 1/5000.
|Flow Cyt||1/40 - 1/100.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
For unpurified use at 1/80.
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).
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 secondary antibody (HRP) (ab131366) as the secondary antibody 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.
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 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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.