Human SARS Coronavirus Spike (Receptor Binding Domain) Insect Cell Lysate (WB positive control)

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Human SARS Coronavirus Spike (Receptor Binding Domain) Insect Cell Lysate (WB positive control): Product Information

Product Description
This SARS Coronavirus spike overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of Coronavirus spike protein (Cat: 40150-V31B2) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Species
SARS
Sequence Information
A DNA sequence encoding the receptor binding domain (RBD) of SARS-CoV (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 SARS-CoV (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.

Human SARS Coronavirus Spike (Receptor Binding Domain) Insect Cell Lysate (WB positive control): Usage Guide

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.

Human SARS Coronavirus Spike (Receptor Binding Domain) Insect Cell Lysate (WB positive control): Synonyms

SARS coronavirus s1 Overexpression Lysate; SARS coronavirus s2 Overexpression Lysate; SARS coronavirus spike Overexpression Lysate; SARS cov spike Overexpression Lysate; SARS ncov RBD Overexpression Lysate; SARS ncov s1 Overexpression Lysate; SARS ncov s2 Overexpression Lysate; SARS ncov spike Overexpression Lysate; SARS novel coronavirus RBD Overexpression Lysate; SARS novel coronavirus s1 Overexpression Lysate; SARS novel coronavirus s2 Overexpression Lysate; SARS novel coronavirus spike Overexpression Lysate; SARS RBD Overexpression Lysate; SARS S1 Overexpression Lysate; SARS s2 Overexpression Lysate; SARS Spike RBD Overexpression Lysate

Coronavirus spike Background Information

The spike (S) glycoprotein of coronaviruses contains protrusions that will only bind to certain receptors on the host cell. Known receptors bind S1 are ACE2, angiotensin-converting enzyme 2; DPP4, dipeptidyl peptidase-4; APN, aminopeptidase N; CEACAM, carcinoembryonic antigen-related cell adhesion molecule 1; Sia, sialic acid; O-ac Sia, O-acetylated sialic acid. The spike is 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. It's been reported that SARS-CoV-2 (COVID-19 coronavirus, 2019-nCoV) can infect the human respiratory epithelial cells through interaction with the human ACE2 receptor. The spike protein is a large type I transmembrane protein containing two subunits, S1 and S2. S1 mainly contains a receptor binding domain (RBD), which is responsible for recognizing the cell surface receptor. S2 contains basic elements needed for the membrane fusion. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity. The main functions for the Spike protein are summarized as: Mediate receptor binding and membrane fusion; Defines the range of the hosts and specificity of the virus; Main component to bind with the neutralizing antibody; Key target for vaccine design; Can be transmitted between different hosts through gene recombination or mutation of the receptor binding domain (RBD), leading to a higher mortality rate.
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.

SARS-CoV-2 Overexpression Lysate

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