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|Human Cell lysate that Human COL2A1 (aa 1242-1487) transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the amino acids (Asp 1242-Leu 1487) of human COL2A1 isoform 1 (P02458-2), known as chondrocalcin, was expressed, with a polyhistidine tag at the N-terminus.|
|The recombinant human COL2A1 (aa 1242-1487) , known as chondrocalcin consists of 265 amino acids and has a calculated molecular mass of 29.8 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of the protein is approximately 37 kDa due to glycosylation.|
|Cell lysate was prepared by homogenization 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.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
COL2A1 is the alpha-1 chain of type II collagen which is a fibrillar collagen found in cartilage and the vitreous humor of the eye. Mutations in this protein are associated with achondrogenesis, chondrodysplasia, early onset familial osteoarthritis, SED congenita, Langer-Saldino achondrogenesis, Kniest dysplasia, Stickler syndrome type I, and spondyloepimetaphyseal dysplasia Strudwick type. In addition, defects in processing chondrocalcin, a calcium binding protein that is the C-propeptide of this collagen molecule, are also associated with chondrodysplasia. There are two transcripts identified for this gene. Type II collagen is specific for cartilaginous tissues. Thus COL2A1 is essential for the normal embryonic development of the skeleton, for linear growth and for the ability of cartilage to resist compressive forces. The regulation of COL2A1, likely results from a balance of both positive and negative proteins. The inhibition of COL2A1 transcription following treatment of chick sternal chondrocytes with growth factors was accompanied by increased EF1 expression. Overexpression of EF1 in differentiated chondrocytes resulted in decreased expression of a reporter construct containing a collagen II promoter/enhancer insert.