|Product Description:||Baculovirus-Insect Cells transfected lysate in which HCoV-EMC/2012 Novel coronavirus (HCoV-EMC/2012) Spike has been over-expressed. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS sample buffer).|
|Preparation Method:||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 with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 minutes in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Lysis Buffer:||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|
|Quality Control Testing:||12.5% SDS-PAGE Stained with Coomassie Blue|
|Stability:||Samples are stable for up to twelve months from date of receipt at -80℃|
|Recommend Usage:||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 boiled for 2-5 min.
3. Store it at -80℃. Recommend to aliquot the cell lysate into smaller quantities for optimal storage. Avoid repeated freeze-thaw cycles.
Notes：The lysate is ready to load on SDS-PAGE for Western blot application. If dissociating conditions are required, add reducing agent prior to heating.|
|Storage Buffer:||In modified RIPA Lysis Buffer|
|Storage Instruction:||Store at -80℃. Aliquot to avoid repeated freezing and thawing|
|Application notes:||WB: Use at an assay dependent dilution.|
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.