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Recombinant full length Human protein
Our Abpromise guarantee covers the use of ab80633 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 - 2 µg/ml. Predicted molecular weight: 18 kDa.|
|IP||Use at 2 µg/mg of lysate. Native verified. Use Protein A.|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|Flow Cyt||Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.|
Lane 1: Wild type DLD-1 whole cell lysate (20 µg)
Lane 2: DLD-1 2,3 DCPE treated knockout DLD-1 whole cell lysate (20 µg)
Lane 3: DLD-1 p21 (KO) control whole cell lysate (20 µg)
Lane 4: DLD-1 p21(KO) 2,3 DCPE treated whole cell lysate (20 µg)
Lane 5: HT1080 whole cell lysate (20 µg)
Lanes 1 - 5: Merged signal (red and green). Green - ab80633 observed at 21 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab80633 was shown to recognize p21 when DLD-1 2,3 DCPE treated knockout samples were used, along with additional cross-reactive bands. Wild-type and DLD-1 2,3 DCPE treated knockout samples were subjected to SDS-PAGE. Ab80633 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/30000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Image from Linley AJ et al, J Biol Chem. 2012 Apr 20;287(17):13633-43. Epub 2012 Mar 5, Fig 3. DOI 10.1074/jbc.M111.308973 April 20, 2012 The Journal of Biological Chemistry, 287, 13633-13643.ab80633 used at a 1/250 dilution for Western Blot.Human cancer cells were collected, washed with 1× PBS, lysed in 1× solution containing 50 mm Tris-HCl (pH 6.8), 100 mm dithiothreitol, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, and 10% (v/v) glycerol and loaded on Tris/glycine SDS-polyacrylamide gels. Proteins were separated alongside a molecular weight marker. Protein bands were transferred onto PVDF membranes. Membranes were blocked with 10% milk/TBS solution with 0.05% Tween 20 containing sodium orthovanadate and sodium fluoride. Following TBS solution with 0.05% Tween 20 washes, membranes were incubated with primary antibodies (in blocking solution) at 4 °C overnight followed by washing and incubation with secondary antibodies for 1 hour at room temperature.
Overlay histogram showing HeLa cells stained with ab80633 (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 (ab80633, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was anti-mouse DyLight® 488 (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). 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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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