Product Description
This SARS CoV spike overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of CoV spike protein (Cat: 40150-V31B2) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Sequence Information
A DNA sequence encoding the receptor binding domain (RBD) of human SARS coronavirus (isolate:WH20) spike (AAX16192.1) (Arg306-Phe527) was expressed with the Fc region of rabbit IgG at the C-terminus.
Molecule Mass
The recombinant receptor binding domain (RBD) of human SARS coronavirus (isolate:WH20) spike comprises 446 amino acids and has a predicted molecular mass of 50.2 kDa. The apparent molecular mass of the protein is approximately 78.9 and 54.3 kDa in SDS-PAGE under reducing conditions.
Preparation Method
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.
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.
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 boil for 2-5 min.
Sample Buffer
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
The spike (S) glycoprotein of coronaviruses contains protrusions that will only bind to certain receptors on the host cell: they are essential for both host specificity and viral infectivity. The term 'peplomer' is typically used to refer to a grouping of heterologous proteins on the virus surface that function together. The spike (S) glycoprotein of coronaviruses is known to be essential in the binding of the virus to the host cell at the advent of the infection process. Most notable is severe acute respiratory syndrome (SARS). The severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein alone can mediate the membrane fusion required for virus entry and cell fusion. It is also a major immunogen and a target for entry inhibitors. The SARS-CoV spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV.
References
Shen S, et al. (2007) Expression, glycosylation, and modification of the spike (S) glycoprotein of SARS CoV. Methods Mol Biol. 379: 127-35. Du L, et al. (2009) The spike protein of SARS-CoV--a target for vaccine and therapeutic development. Nat Rev Microbiol. 7 (3): 226-36. Xiao X, et al. (2004) The SARS-CoV S glycoprotein. Cell Mol Life Sci. 61 (19-20): 2428-30. 
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