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Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human Calreticulin.
(Peptide available as ab39896.)
Our Abpromise guarantee covers the use of ab39897 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 1 µg/ml.|
|WB||1/250. Detects a band of approximately 55 kDa (predicted molecular weight: 48 kDa).|
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Calreticulin knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: NIH3T3 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab39897 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab39897 was shown to recognize Calreticulin when Calreticulin knockout samples were used, along with additional cross-reactive bands. Wild-type and Calreticulin knockout samples were subjected to SDS-PAGE. ab39897 and ab8245 (loading control to GAPDH) were diluted 1/250 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
ICC/IF image of ab39897 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab39897, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
The predicted band size is 48kDa based on Swiss-prot data, however a band of 55kDa is consistent with that observed in other commercially available antibodies to Calreticulin. This difference in size is likely to be the result of post-translational modification.
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