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Recombinant full length Human protein
Abcam is committed to meeting high standards of ethical manufacturing and has decided to discontinue this product by June 2019 as it has been generated by the ascites method. We are sorry for any inconvenience this may cause. We would recommend antibody ab109520 as a replacement.
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 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 specifically react with p21 in DLD-1 cells in the presence of 2,3 DCPE treatment. No band was observed in knockout samples +/-2,3 DCPE treatment. 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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