Confocal immunofluorescent analysis of MCF-7 cells using Hexokinase I (C35C4) Rabbit mAb (Alexa Fluor® 647 Conjugate) (blue pseudocolor) and MitoTracker® Red CMXRos (red) showing colocalization with mitochondria. Nuclei have been labeled with p53 (1C12) Mouse mAb (Alexa Fluor® 488 Conjugate) #2015 (green).
Flow cytometric analysis of MCF-7 cells using Hexokinase I (C35C4) Rabbit mAb (Alexa Fluor® 647 Conjugate) (green) compared to XP® Rabbit (DA1E) mAb IgG Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (red).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry and immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Hexokinase I (C35C4) Rabbit mAb #2024.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised December 2010
Protocol Id: 220
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Hexokinase I (C35C4) Rabbit mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of total hexokinase I protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence of human hexokinase I. This antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.
Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate, the first step in glycolysis. Four distinct mammalian hexokinase isoforms, designated as hexokinase I, II, III, and IV (glucokinase), have been identified. Hexokinases I, II, and III are associated with the outer mitochondrial membrane and are critical for maintaining an elevated rate of aerobic glycolysis in cancer cells (Warburg Effect) (1) in order to compensate for the increased energy demands associated with rapid cell growth and proliferation (2,3).
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