Alexa Fluor® 647 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB197864)

Flow cytometry scatter plots showing human peripheral mononuclear cells (PBMCs), either stained with ab197864 (right) or isotype control (left). The cells were fixed and permeabilised using BD CytoFix/CytoPerm™ (20 min). The cells were incubated in 1x PBS containing 10 μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab197864) (1x 10⁶ in 100μl at 0.2 μg/ml (1/2500)) for 30min on ice. Isotype control antibody was Alexa Fluor® 647 Rabbit IgG, monoclonal [EPR25A] - Isotype Control. The cells were simultaneously stained with CD3.

Acquisition of >30000 events were collected using a 40 mW Red laser (638nm) and 660/10 bandpass filter.

This data was acquired using a Beckman Coulter CytoFlexLX flow cytometer.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB197864)

Flow cytometry overlay histogram showing wild-type HEK293T (green line) and HK1 knockout HEK293T stained with ab197864 (magenta line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab197864) (1x 10⁶ in 100μl at 5 μg/ml (1/100)) for 30min at 22°C.

Isotype control antibody was Alexa Fluor® 647 Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) in HEK293T WT cells (black line) and HEK293T-HK1 KO cells (grey line), used at the same concentration and conditions as the primary antibody.

Acquisition of >5000 events were collected using a 40 mW Red laser (638nm) and 660/10 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB197864)

ab197864 staining Hexokinase 1 in HCT116 cells. The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab at a 1/50 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 647 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB197864)

Immunofluorescent analysis of 4% PFA-fixed 0.1% Triton X-100 permeabilized HEK-293 WT and HEK-293-HK1 KO cells labelling Hexokinase 1 with ab197864 at 0.2 μg/ml concentration (Magenta). Cells were counterstained with ab195887 Anti-Tubulin (Alexa Fluor® 488) at 1 : 250 (2 μg/ml) (Green). The nuclear counterstain was DAPI (Blue).