Western blot analysis of extracts from mock-infected Vero-E6 cells (lane 1) and SARS-CoV-2-infected Vero-E6 cells (lane 2), using SARS-CoV-2 Spike Protein (S1-NTD) Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The antibody detects full-length (uncleaved) SARS-CoV-2 spike protein, and the fragment corresponding to the S1 domain generated by endogenous protease cleavage. Vero-E6 cells courtesy of Dr. Mohsan Saeed, National Emerging Infectious Diseases Laboratories, Boston University.
Western blot analysis of SARS-CoV-2 Spike S1-NTD (16-316) Recombinant Protein (8xHis-Tag) #88587 (lane 1), SARS-CoV-2 Spike (trimeric) (16-1208) Recombinant Protein (8xHis-Tag) #65444 (lane 2), SARS-CoV-2 Spike RBD (318-541) Recombinant Protein (8xHis-Tag) #48801 (lane 3), or SARS-CoV-2 Spike RBD (multimeric) (319-591) Recombinant Protein (8xHis-Tag) #17862 (lane 4), using SARS-CoV-2 Spike Protein (S1-NTD) Antibody (upper) and His-Tag (D3I1O) XP® Rabbit mAb #12698 (lower). Due to the location of the epitope (surrounding Pro25 of SARS-CoV-2 spike protein), the antibody detects recombinant proteins corresponding to the full-length ectodomain and the S1-NTD of SARS CoV-2 spike protein but does not detect recombinant proteins corresponding only to the receptor binding domain (RBD).
Western blot analysis of extracts from 293T cells, mock transfected (lane 1) or transiently transfected with expression constructs encoding Myc/DDK-tagged SARS-CoV-2 spike (lane 2), Myc/DDK-tagged SARS-CoV spike (lane 3), or Myc/DDK-tagged MERS-CoV spike (lane 4), using SARS-CoV-2 Spike Protein (S1-NTD) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The antibody detects full-length (uncleaved) SARS-CoV-2 spike protein, and the fragment corresponding to the S1 domain generated by endogenous protease cleavage. Proteolytic cleavage of SARS-CoV spike is not observed in these experimental conditions (transient transfection).
Immunoprecipitation of SARS-CoV-2 spike protein from extracts of 293T cells transiently transfected with an expression construct encoding SARS-CoV-2 spike protein. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is SARS-CoV-2 Spike Protein (S1-NTD) Antibody. Western blot analysis was performed using SARS-CoV-2 Spike Protein (S1-NTD) (E7M5X) Mouse mAb #42172. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.
|MW (kDa)||110, 220|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 410
SARS-CoV-2 Spike Protein (S1-NTD) Antibody recognizes endogenous levels of total SARS-CoV-2 spike protein. This antibody detects full-length protein, and also detects the S1 fragment generated by furin cleavage. It does not cross-react with spike proteins from SARS or MERS coronaviruses.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro25 of SARS-CoV-2 spike protein. Antibodies are purified by peptide affinity chromatography.
The cause of the COVID-19 pandemic is a novel and highly pathogenic coronavirus, termed SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2). SARS-CoV-2 is a member of the Coronaviridae family of viruses (1). The genome of SARS-CoV-2 is similar to other coronaviruses, and is comprised of four key structural proteins: S, the spike protein, E, the envelope protein, M, the membrane protein, and N, the nucleocapsid protein (2). Coronavirus spike proteins are class I fusion proteins and harbor an ectodomain, a transmembrane domain, and an intracellular tail (3,4). The highly glycosylated ectodomain projects from the viral envelope surface and facilitates attachment and fusion with the host cell plasma membrane. The ectodomain can be further subdivided into host receptor-binding domain (RBD) (S1) and membrane-fusion (S2) subunits, which are produced upon proteolysis by host proteases at S1/S2 and S2’ sites. S1 and S2 subunits remain associated after cleavage and assemble into crown-like homotrimers (2,4). In humans, both SARS-CoV and SARS-CoV-2 spike proteins utilize the angiotensin-converting enzyme 2 (ACE2) protein as a receptor for cellular entry (5-7). Spike protein subunits represent a key antigenic feature of coronavirus virions, and therefore represent an important target of vaccines, novel therapeutic antibodies, and small-molecule inhibitors (8,9).
Explore pathways + proteins related to this product.
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