Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification. MGP is mainly synthesized and expressed in cartilage and the vessel wall, where it is produced by the chondrocytes and vascular smooth muscle cells, respectively. During biosynthesis, MGP undergoes two forms of post-translational modifications: the phosphorylation of 3 serine residues and the γ-carboxylation of 5 glutamate residues. Phosphorylation of MGP is essential for its regular cellular secretion, and the vitamin K-dependent carboxylation is crucial for the calcification inhibitory activity. MGP plays a role in bone metabolism and atherosclerosis.
Immunohistochemistry Protocol Staining of paraffin embedded tissue sections fixed with 4.5% neutrally buffered formalin:1. Deparaffinize and rehydrate tissue sections (100% xylol 2x; 100% ethanol; 96% ethanol; 70% ethanol; 50% ethanol). 2. Wash sections in TBST. 3. Heat 0.2% citric acid, pH 6.0, in microwave until boiling. 4. Put sections in pre-warmed citric acid and "keep-warm" in the microwave for 15 min.5. Cool down sections for 15 min. and wash three times in TBST. 6. Apply MAb to Matrix Gla Protein (52.1C5D) (Prod. No. ALX-804-512) (1:200) in TBST containing 1% BSA and incubate overnight at +4°C.7. Wash sections 3x in TBST. 8. Use appropriate ABC detection kit (e.g. VECTASTAIN ABC Kit) according to manufacturer’s instructions. 9. Apply and stain sections with substrate. 10. Wash sections in tap water for 10 min. 11. Counterstain sections with haemalaun (20 sec.). 12. Wash sections in tap water for 10 min. 13. Coverslip section using an aqueous mounting media.Western Blot Recommendations 1. Block membrane with PBST containing 3% BSA for at least 1 hour at room temperature.2. Wash 3x in PBST. 3. Incubate overnight at +4°C with MAb to Matrix Gla Protein (52.1C5D) (Prod. No. ALX-804-512) (1:500) in PBST containing 1% non-fat dried milk.
