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Our Abpromise guarantee covers the use of ab58301 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 - 5 µg/ml. Predicted molecular weight: 62 kDa.|
|IHC-P||Use a concentration of 3 µg/ml.|
|ICC/IF||Use a concentration of 1 µg/ml.|
Lanes 1, 3 and 5: PKR knockout HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: Wild-type HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target - ab58301 observed at 62 kDa
Lanes 3 and 4: Red signal from loading control - ab181602 observed at 37 kDa
Lanes 5 and 6: Merged (red and green) signal
ab58301 was shown to specifically react with PKR when PKR knockout samples were used. Wild-type and PKR knockout samples were subjected to SDS-PAGE. ab58301 and ab181602 (loading control to GAPDH) were diluted to 1 µg/mL and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
ab58301 staining PKR in wild-type HAP1 cells (top panel) and EIF2AK2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab58301 at 1μg/ml concentration and ab6046 at 1μg/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to Rabbit IgG (Alexa Fluor® 594) (ab150084) at 2 μg/ml (showed in pseudocolour red) . Nuclear DNA was labelled in blue with DAPI.
ab58301 at 10 ug/ml staining PKR in human HeLa cells by Immunocytochemistry/ Immunofluorescence.