Mouse Carbonic Anhydrase XIV HEK293 Overexpression Lysate: 产品信息
This Mouse Carbonic Anhydrase XIV overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Carbonic Anhydrase XIV protein (Cat: 50013-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse CA14 (NP_035927.1) (Met 1-Met 290) was expressed with a C-terminal polyhistidine tag.
The recombinant mouse CA14 consists of 286 amino acids and has a calculated molecular mass of 32.2 kDa. As a result of glycosylation, the recombinant protein migrates as an approximately 45-48 kDa protein in SDS-PAGE under reducing conditions.
Mouse Carbonic Anhydrase XIV HEK293 Overexpression Lysate: 使用指南
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
The carbonic anhydrases (or carbonate dehydratases) are classified as metalloenzyme for its zinc ion prosthetic group and form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons, a reversible reaction that takes part in maintaining acid-base balance in blood and other tissues. The carbonic anhydrasekl (CA) family consists of at least 11 enzymatically active members and a few inactive homologous proteins. CAXIV is a member of CA family that showed an overall similarity of 29–46% to other active CA isozymes. The highest percentage similarity was with a transmembrane CA isoform, CAXII. The CAXIV was found high concentrations in human heart, brain, liver, and skeletal muscle but lower in the colon, small intestine, urinary bladder, and kidney. No CAXIV mRNA was seen in the salivary gland and pancreas. CAXIV is a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.
carbonic anhydrase XIV
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Parkkila S, et al. (2001) Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain. PNAS. 98(4): 1918-23.