The monoclonal antibody CK-BYK/21E10 recognizes human creatine kinase B-type, also known as B-CK. Human B-CK is a protein of 381 amino acids (~45 kDa), expressed in a number of tissues. B-CK is most abundant in adult brain, approx. 5-fold lower in the stomach, 10-fold lower in the heart and barely detectable in liver. In brain, whereas most B-CK has been shown to be cytosolic, several of the reactions requiring B-CK are membrane-associated. B-CK belongs to the creatine kinase (CK) isoenzymes that catalyse the synthesis of phosphocreatine (PCr) and its subsequent use in the regeneration of ATP in cell types where the consumption of ATP is rapid and/or sudden. In the brain the different CK isoforms constitute an energy shuttle wherein ATP produced in the mitochondria is used by a mitochondrial CK [e.g. ubiquitous mitochondrial creatine kinase (uMi-CK)] to generate PCr, which is then transported and used by a cytoplasmic CK [e.g. brain creatine kinase (B-CK)] to regenerate ATP at discrete cellular sites of high ATP turnover. B-CK appears to have a role in regenerating ATP needed for the transport of ions and neurotransmitters since CKB has been localized to brain synaptic plasma membranes, possibly coupled to Na+/K+-ATPase and acetylcholine receptor-rich membranes.
Expression of B-CK is developmentally controlled: in rat, brain CK protein at birth is extremely low and increases 10-fold until week 4. This reflects the many energy-demanding processes in brain during brain development.
Frozen sections, Immuno fluorescence, Paraffin sections, Western blot
W: reduced; size ~43-46 kDa (Ref 1-2).
IHC-F: fixed 2 hr in 2% paraformaldehyde and 0.1 % glutaraldehyde in 0.1 M PB; stored until use in 1 % paraformaldehyde; blocking with 1% normal swine serum (Ref 1).
IHC-P: antigen retrieval: 0.5 % pepsin in 0.01 N HCl for 15 min at 37 °C; endogenous peroxidise blocking by using 3 % H2O2 in PBS for 30 min; blocking with 1% normal swine serum for 30 min. Purkinje cells serve as positive control and connective tissue as negative control (Ref 1).
Dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.
Brain lysate, Human Purkinje cells