The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 71 kDa (predicted molecular weight: 71 kDa).
Use at an assay dependent concentration.
Catalyzes the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP), the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle.
Carbohydrate biosynthesis; gluconeogenesis.
Involvement in disease
Defects in PCK2 are the cause of mitochondrial phosphoenolpyruvate carboxykinase deficiency (M-PEPCKD) [MIM:261650]. A metabolic disorder resulting from impaired gluconeogenesis. It is a rare disease with less than 10 cases reported in the literature. Clinical characteristics include hypotonia, hepatomegaly, failure to thrive, lactic acidosis and hypoglycemia. Autoposy reveals fatty infiltration of both the liver and kidneys. The disorder is transmitted as an autosomal recessive trait.
Belongs to the phosphoenolpyruvate carboxykinase [GTP] family.
Phosphorylated upon DNA damage, probably by ATM or ATR.
PCK2 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to PCK2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab70359. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 71kDa: PCK2.
ICC/IF image of ab70359 stained MCF7 cells. The cells were 100% methanol fixed (5 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 (ab70359, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa, HepG2 and Hek293 cells at 5µg/ml.
IHC image of PCK2 staining in normal human liver formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab70659, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
Méndez-Lucas A et al. Mitochondrial PEPCK is a Pro-Survival, ER-Stress Response Gene Involved in Tumor Cell Adaptation to Nutrient Availability. J Biol ChemN/A:N/A (2014).
Read more (PubMed: 24973213) »
Méndez-Lucas A et al. PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis. J Hepatol59:105-13 (2013).
Mouse, Rat, Rabbit, Human
Read more (PubMed: 23466304) »