GCKR Antibody Staining Protocol for Immunohistochemistry


Description: Glucokinase (also designated hexokinase IV or GCK) plays a key role in the regulation of glucose-induced insulin secretion. GCK is expressed in pancreatic beta cells, where it functions as a glucose sensor, determining the “set point” for insulin secretion. GCK is also expressed in the liver, where it catalyzes the first step in the disposal of glucose. A lack of glucokinase activity leads to reduced insulin secretion and hyperglycemia and has been implicated as a cause for maturity onset diabetes of the youth (MODY). Heterozygous point mutations in the gene encoding GCK have been detected in individuals suffering from MODY. GCK is regulated by GCKR (glucokinase regulatory protein). GCKR is a 68 kDa protein which is expressed in pancreatic beta cells and in the liver.


Primary Antibody

Name: GCKR (N-19) Antibody

Clone: Goat polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-6340

Dilution: 1:50 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin sections
Positive Control: Pancreas, liver
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary