Human Collagen II/COL2A1 HEK293 Overexpression Lysate: Product Information
This Human Collagen II/COL2A1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Collagen II/COL2A1 protein (Cat: 11790-H07H) from the overexpression lysate was verified.
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.
Human Collagen II/COL2A1 HEK293 Overexpression Lysate: Usage Guide
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.
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.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human Collagen II/COL2A1 HEK293 Overexpression Lysate: Alternative Names
Human ANFH Overexpression Lysate; Human AOM Overexpression Lysate; Human COL11A3 Overexpression Lysate; Human SEDC Overexpression Lysate; Human STL1 Overexpression Lysate
Collagen II/COL2A1 Background Information
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.
collagen, type II, alpha 1
Chan D, et al. (1995) A COL2A1 mutation in achondrogenesis type II results in the replacement of type II collagen by type I and III collagens in cartilage. J Biol Chem. 270 (4): 1747-53.
Donoso LA, et al. (2003) Clinical variability of Stickler syndrome: role of exon 2 of the collagen COL2A1 gene. Surv Ophthalmol. 48 (2): 191-203.
Meulenbelt I, et al. (1999) Haplotype analysis of three polymorphisms of the COL2A1 gene and associations with generalised radiological osteoarthritis. Ann Hum Genet. 63 (5): 393-400.
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