Revision 5
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IHC-P, IF-IC

REACTIVITY:

Vir

SENSITIVITY:

Endogenous

MW (kDa):

110, 220

Source/Isotype:

Rabbit IgG

UniProt ID:

#P0DTC2

Entrez-Gene Id:

43740568

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunohistochemistry (Paraffin) 1:200 - 1:800
Immunofluorescence (Immunocytochemistry) 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb recognizes endogenous levels of total SARS-CoV-2 spike protein. The antibody detects full-length SARS-CoV-2 spike protein and also detects the S1 domain generated by furin cleavage. The antibody has been confirmed to detect the following SARS-CoV-2 isolates and variants: USA-WA1/2020, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). The antibody does not cross-react with spike proteins from SARS or MERS coronaviruses. Non-specific nuclear foci staining was observed in some mouse tissues by immunohistochemistry.

Species Reactivity:

Virus

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein corresponding to the S1 domain of SARS-CoV-2 spike protein (USA-WA1/2020 isolate).

Background

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).

  1. Zhou, P. et al. (2020) Nature 579, 270-3.
  2. Tortorici, M.A. and Veesler, D. (2019) Adv Virus Res 105, 93-116.
  3. Li, F. et al. (2006) J Virol 80, 6794-800.
  4. Li, F. (2016) Annu Rev Virol 3, 237-61.
  5. Shang, J. et al. (2020) Nature 581, 221-4.
  6. Wrapp, D. et al. (2020) Science 367, 1260-3.
  7. Yan, R. et al. (2020) Science 367, 1444-8.
  8. Yuan, Y. et al. (2017) Nat Commun 8, 15092.
  9. Amanat, F. and Krammer, F. (2020) Immunity 52, 583-9.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: 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.

Applications Key

WB: Western Blotting IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a registered trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Revision 5
#99423

SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb

Western Blotting Image 1: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (lane 1) or transiently transfected with plasmid encoding Myc/DDK-tagged SARS-CoV-2 spike protein (lane 2), using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb (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.
Western Blotting Image 2: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
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) (E5S3V) Rabbit mAb (upper) and His-Tag (D3I1O) XP® Rabbit mAb #12698 (lower). Due to the location of the epitope (within the S1 domain 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 protein corresponding only to the receptor binding domain (RBD).
Western Blotting Image 3: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Western blot analysis of extracts from mock-infected Vero-E6 cells (lane 1), SARS-CoV-2-infected Vero-E6 cells (lane 2), and isolated SARS-CoV-2 virions (lane 3), using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb (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. SARS-CoV-2 virions and infected Vero-E6 cells courtesy of Dr. Mohsan Saeed, National Emerging Infectious Diseases Laboratories, Boston University.
Immunohistochemistry Image 1: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2 positive human placenta (left, positive) and normal human placenta (right, negative) using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb. 
Immunohistochemistry Image 2: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2-infected hACE2 K18 mouse lung using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). Human ACE2 transgenic mouse lung tissue was generously provided by Dr. Nicholas Crossland and Dr. Florian Douam, National Emerging Infectious Diseases Laboratories, Boston University.
Immunohistochemistry Image 3: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 293T cell pellet transfected with SARS-CoV-2 spike protein (left-top) and various paraffin-embedded human tissues: colon adenocarcinoma (right-top), prostate adenocarcinoma (right-bottom), and salivary gland small cell carcinoma (left-bottom) using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb. Note the lack of staining in the tissues, all of which were procured prior to 2019, and therefore serve as reliable negative controls.
Immunohistochemistry Image 4: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 293T cell pellet, untransfected (left) or SARS-CoV-2 spike protein transfected (right), using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb.
Immunohistochemistry Image 5: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded A549-hACE2-hTMPRSS2 cell pellet, mock infected or infected with SARS-CoV-2 2019-nCoV/USA-WA1/2020 variant, SARS-CoV-2 Alpha (B.1.1.7) variant, SARS-CoV-2 Beta (B.1.351) variant (from top left to right as indicated), SARS-CoV-2 Gamma (P.1) variant, SARS-CoV-2 Delta (B.1.617.2) variant, or SARS-CoV-2 Omicron (B.1.1.529) variant (from bottom left to right as indicated), using SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb. A549-hACE2-hTMPRSS2 cell pellets were generously provided by Dr. Nicholas Crossland and Dr. Florian Douam, National Emerging Infectious Diseases Laboratories, Boston University.
Immunofluorescence Image 1: SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HCT 116 cells transiently transfected with SARS-CoV-2 spike protein, labeled with SARS-CoV-2 Spike Protein (S1) (E5S3V) Rabbit mAb (left, green) and co-labeled with DyLight 650 Phalloidin #12956 (right, red) and DAPI #4083 (right, blue).