This Human CYTL1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CYTL1 protein (Cat: 13647-H05H) from the overexpression lysate was verified.
A DNA sequence encoding the human CYTL1 (NP_061129.1) (Met1-Arg136) was expressed with the Fc region of mouse IgG1 at the C-terminus.
The recombinant human CYTL1 consists 348 amino acids and predicts a molecular mass of 39.8 kDa.
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.
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.
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.
Cytokine-like protein 1 (CYTL1) is a small widely expressed secreted protein lacking significant primary sequence homology to any other known protein. Cytokine-like 1 (CYTL1) is a novel potential cytokine that was first identified in CD34(+) cells derived from bone marrow and cord blood, and it was also found using our immunogenomics strategy. CYTL1 expression appears to be highest in the hematopoietic system and in chondrocytes; however, maintenance of cartilage in mouse models of arthritis is its only reported function in vivo. Despite lacking sequence homology to chemokines, CYTL1is predicted by computational methods to fold like a chemokine, and has been reported to function as a chemotactic agonist at the chemokine receptor CCR2 in mouse monocyte/macrophages. Recombinant CYTL1 promoted calcium flux in chondrocytes. Cytokine-like protein 1 (Cytl1), had been described as a protein expressed in CD34+ cells, and as a functional secreted protein involved in chondrogenesis and cartilage development.