Alexa Fluor® 488 Anti-Hexokinase 1 抗体 [EPR10134(B)] - Mitochondrial Outer Membrane Marker

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 488 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB184818)

Flow cytometry scatter plots showing human peripheral mononuclear cells (PBMCs), either stained with ab184818 (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 (ab184818) (1x 10⁶ in 100μl at 0.2 μg/ml (1/2500)) for 30min on ice. Isotype control antibody was Recombinant Alexa Fluor® 488 Rabbit IgG, monoclonal [EPR25A] - Isotype Control. The cells were simultaneously stained with CD3.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 488 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB184818)

Flow cytometry overlay histogram showing wild-type HEK293T (green line) and HK1 knockout HEK293T stained with ab184818 (magenta line). The cells were fixed with 80% methanol (5 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 (ab184818) (1x 10⁶ in 100μl at 1 μg/ml (1/500)) for 30min at 22°C.

Isotype control antibody was Recombinant Alexa Fluor® 488 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 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in wild-type HEK293T fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 488 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB184818)

ab184818 staining Hexokinase 1 in HeLa cells. The cells were fixed with 4% PFA (10min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab184818 at a working dilution of 1 in 100 overnight at +4°C (shown in green). AlexaFluor^® 350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Alexa Fluor® 488 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB184818)

Overlay histogram showing HeLa cells stained with ab184818 (red line). The cells were fixed with 4% formaldehyde (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 (ab184818, 1/50 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 488 used at the same concentration and conditions as the primary antibody. 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 fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Alexa Fluor® 488 Anti-Hexokinase 1 antibody [EPR10134(B)] - Mitochondrial Outer Membrane Marker (AB184818)

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 ab184818 at 0.2 μg/ml concentration (Green). Cells were counterstained with ab190573 Anti-Tubulin (Alexa Fluor® 647) at 1 : 250 (2 μg/ml) (Magenta). The nuclear counterstain was DAPI (Blue).