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|Human Cell lysate that Cynomolgus CD3D & CD3E Heterodimer 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 Cynomolgus (Macaca fascicularis) CD3E extracellular domain (Met 1-Asp 117) was fused with the C-terminal his-tagged Fc region of human IgG1 at the C-terminus, constructed the plasmid 1; A DNA sequence encoding the Cynomolgus (Macaca fascicularis) CD3D extracellular domain (Met 1-Ala 105) with the C-terminal flag-tagged Fc region of human IgG1 at the C-terminus, constructed the plasmid 2. The two plasmids were co-expressed and the CD3E&CD3D heterodimer was purified.|
|Gln 22 & Phe 22|
|The recombinant heterodimer of cynomolgus CD3E&CD3D comprises 679 (346+333) amino acids and has a calculated molecular mass of 76.7 (39.1+ 37.6) KDa. The apparent molecular mass of cyno CD3E&CD3D heterodimer is approximately 52 & 43 KDa respectively in SDS-PAGE.|
|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.