Overview

Applications

Our Abpromise guarantee covers the use of ab70359 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 71 kDa (predicted molecular weight: 71 kDa).Can be blocked with Human PCK2 peptide (ab87120).
IP Use at an assay dependent concentration.

Target

  • FunctionCatalyzes 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.
  • PathwayCarbohydrate biosynthesis; gluconeogenesis.
  • Involvement in diseaseDefects 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.
  • Sequence similaritiesBelongs to the phosphoenolpyruvate carboxykinase [GTP] family.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localizationMitochondrion.
  • Information by UniProt
  • Database links
  • Alternative names
    • EC 4.1.1.32 antibody
    • GTP mitochondrial precursor antibody
    • HGNC:8725 antibody
    • mitochondrial antibody
    • Mitochondrial phosphoenolpyruvate carboxykinase 2 antibody
    • OTTHUMP00000164700 antibody
    • PCK2 antibody
    • PCKGM_HUMAN antibody
    • PE antibody
    • PEP carboxykinase antibody
    • PEPCK antibody
    • PEPCK deficiency mitochondrial antibody
    • PEPCK M antibody
    • PEPCK-M antibody
    • PEPCK2 antibody
    • Phosphoenolpyruvate carboxykinase [GTP] antibody
    • Phosphoenolpyruvate carboxykinase 2 (mitochondrial) antibody
    • Phosphoenolpyruvate carboxykinase 2 mitochondrial antibody
    • Phosphoenolpyruvate carboxylase antibody
    • Phosphopyruvate carboxylase antibody
    see all

Anti-PCK2 antibody images

  • Anti-PCK2 antibody (ab70359) at 1 µg/ml + HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 71 kDa
    Observed band size : 71 kDa
    Additional bands at : 40 kDa,50 kDa. We are unsure as to the identity of these extra bands.
  • 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.

References for Anti-PCK2 antibody (ab70359)

This product has been referenced in:
  • 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 Chem N/A:N/A (2014). WB, IHC-P ; Human . Read more (PubMed: 24973213) »
  • Méndez-Lucas A  et al. PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis. J Hepatol 59:105-13 (2013). WB, ICC/IF ; Mouse, Rat, Rabbit, Human . Read more (PubMed: 23466304) »

See all 2 Publications for this product

Product Wall

Application Western blot
Sample Rat Tissue lysate - whole (Liver)
Loading amount 15 µg
Specification Liver
Gel Running Conditions Reduced Denaturing (4-12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Rajesh

Verified customer

Submitted Mar 09 2011

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"