Detection of Human Hexokinase 1/2 by Western Blot.
Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, K562 human chronic myelogenous leukemia cell line, and HEK293 human embryonic kidney cell line. PVDF membrane was probed with 1:1000 dilution of Rabbit Anti-Human Hexokinase 1/2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8178) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Hexokinase 1/2 at approximately 105 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Hexokinase 1/2 in Human Brain Medulla Tissue.
Hexokinase 1/2 was detected in immersion fixed paraffin-embedded sections of human brain medulla tissue using Rabbit Anti-Human Hexokinase 1/2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF8178) at 1:200 dilution overnight at 4 °C. Tissue was stained using the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). Specific staining was localized to neurons. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Preparation and Storage
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C, as supplied.
1 month, 2 to 8 °C under sterile conditions after opening.
6 months, -20 to -70 °C under sterile conditions after opening.
Background: Hexokinase 1/2
Hexokinases phosphorylate hexose to form hexose 6-phosphate, the first step in hexose metabolism (1). Phosphorylation of a hexose adds charge to molecule thereby making it difficult to transport out of a cell. The hexose is therefore retained for intracellular metabolic processes, such as glycolysis or glycogen synthesis. In most organisms, glucose is the most important substrate of hexokinases and glucose-6-phosphate is the most important product. There are four mammalian hexokinases (2). Hexokinase 1, 2 and 3 are referred to as high-affinity hexokinases because their Km for glucose is below 1 mM. Hexokinase 4 is specific for glucose and is also referred to as glucokinase (3). Hexokinase I localizes to the outer membrane of mitochondria and is found in all mammalian tissues. The amino acids involved in mitochondrion membrane localization (4) have been removed in the recombinant enzyme. Hexokinase 1 (HK1) contains two homologous halves that are believed to be evolved from a single ancestral hexokinase by gene duplication and fusion (5). While the regulatory function is associated with the N-terminal half, the catalytic site is associated with the C-terminal half. HK1 is insensitive to product inhibition (6). Mutation in the active site of human hexokinase is associated with hexokinase deficiency and severe nonspherocytic hemolytic anemia (7). The enzymatic activity of recombinant human HK1 is measured using a phosphatase-coupled method (8).
1. Aleshin, A.E. et al. (1998) Structure 6:39-50 2. Takeda, J. et al. (1993) J. Biol. Chem. 268:15200. 3. Lange, A.J. et al. (1991) Biochem. J. 277:159–163. 4. Magnani, M. et al., J. Biol. Chem. 266: 502. 5. Nishi, S. et al. (1988) Biochem. Biophys. Res. Commun. 157:937. 6. Magnani, M. et al. (1992) Biochem. J. 285:193. 7. Van Wijk R. et al. (2003) Blood 101:345. 8. Wu, Z.L. (2011) PLoS One 6:e23172.
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