The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 87 kDa.
Use a concentration of 5 µg/ml.
Use 0.01-0.1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration. Detection limit for recombinant tagged Mark3 is approximately 3ng/ml when used as a capture antibody.
FunctionInvolved in the specific phosphorylation of microtubule-associated proteins for tau, MAP2 and MAP4. Phosphorylates CDC25C on 'Ser-216'.
Sequence similaritiesBelongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. MARK subfamily. Contains 1 KA1 (kinase-associated) domain. Contains 1 protein kinase domain. Contains 1 UBA domain.
Overlay histogram showing HeLa cells stained with ab77199 (red line). The cells were fixed with 80% methanol (5 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 (ab77199, 0.01μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. 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 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
Western blot - Mark3 antibody (ab77199)
Anti-Mark3 antibody (ab77199) at 5 µg/ml + Recombinant tagged human Mark3 fragment at 0.2 µg
Secondary Goat Anti-Mouse IgG (H&L)-HRP Conjugate at 1/5000 dilution
ICC/IF image of ab77199 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab77199, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
References for Anti-Mark3 antibody (ab77199)
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