Anti-alpha Adaptin antibody [AP6] (ab2730)

Immunocytochemistry/ Immunofluorescence-alpha Adaptin antibody [AP6](ab2730)

ICC/IF image of ab2730 stained MCF7 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 (ab2730, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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.

Flow Cytometry-alpha Adaptin antibody [AP6](ab2730)

Overlay histogram showing MCF7 cells stained with ab2730 (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 (ab2730, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Western blot - Anti-alpha Adaptin antibody [AP6] (ab2730)

ab2730 used in Western Blot at a 1/1000 dilution.

Sucrose density gradient fractionation of detergent resistant cell lysates from WT and DHHC5-GT murine neuronal stem cells. The isolation of detergent-resistant membranes was performed according to a previously published protocol. NSCs growing in growth medium (∼300 μl of packed cells) were harvested and washed three times with ice-cold PBS. The cells were gently resuspended by pipetting up and down in 700 μl of 1% Triton X-100 in HEPES buffer (25 mm HEPES-HCl, pH 6.5, 150 mm NaCl, 1 mm EDTA, and protease inhibitor mixture), homogenized using a Teflon-coated Dounce homogenizer (30 strokes), then incubated on ice for 30 min, and fractionated on a sucrose density gradient. The fractions were collected from top (fraction 1) to bottom (fraction 13) and analyzed by immunoblotting. The results shown were from one of two independent experiments giving similar results. α-Adaptin is a marker for clathrin-coated vesicles.

Image from Li Y et al, J Biol Chem. 2012 Jan 2;287(1):523-30. Epub 2011 Nov 11, Fig 2. DOI 10.1074/jbc.M111.306183 January 2, 2012 The Journal of Biological Chemistry, 287, 523-530.