Human CKB is a protein of 381aa (~45kD), expressed in a number of tissues. CKB is most abundant in adult brain, ~5-fold lower in the stomach, 10-fold lower in the heart and barely detectable in liver. In brain, whereas most CKB has been shown to be cytosolic, several of the reactions requiring CKB are membrane-associated. CKB 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 (CKB)] to regenerate ATP at discrete cellular sites of high ATP turnover. CKB 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 CKB is developmentally controlled: in rat, brain CKB 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.
Applications:
Suitable for use in Western Blot, Immunofluorescence and Immunohistochemistry. Other applications not tested.
Recommended Dilution:
Western Blot: 1:50 (reduced; size ~43-46kD)
Immunohistochemistry (Frozen): fixed 2hr in 2% paraformaldehyde and 0.1 % glutaraldehyde in 0.1M PB; stored until use in 1 % paraformaldehyde; blocking with 1% normal swine serum
Immunohistochemistry (Paraffin): antigen retrieval: 0.5% pepsin in 0.01N HCl for 15min at 37 degrees C; endogenous peroxidise blocking by using 3% H2O2 in PBS for 30min; blocking with 1% normal swine serum for 30min.; Purkinje cells serve as positive control and connective tissue as negative control
Optimal dilutions to be determined by the researcher.
Positive Control: Human Purkinje cells
Storage and Stability:
May be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. Aliquots are stable for at least 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
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