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38559
MERS-CoV Spike Protein (E9P2L) Mouse mAb
Primary Antibodies
Monoclonal Antibody

MERS-CoV Spike Protein (E9P2L) Mouse mAb #38559

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  1. WB
Western Blotting Image 1: MERS-CoV Spike Protein (E9P2L) Mouse mAb
Western blot analysis of extracts from 293T cells, mock transfected (lane 1) or transiently transfected with expression constructs encoding Myc/DDK-tagged MERS-CoV spike (lane 2), Myc/DDK-tagged SARS-CoV spike (lane 3), or Myc/DDK-tagged SARS-CoV-2 spike (lane 4), using MERS-CoV Spike Protein (E9P2L) Mouse mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The antibody detects full-length (uncleaved) MERS-CoV spike protein, and the fragment corresponding to the S1 domain generated by endogenous protease cleavage. Proteolytic cleavage of SARS-CoV spike protein is not observed in these experimental conditions (transient transfection).
To Purchase # 38559
Cat. # Size Qty. Price
38559S
100 µl $ 287

Supporting Data

REACTIVITY Vir
SENSITIVITY Endogenous
MW (kDa) 200, 120
Source/Isotype Mouse IgG1 kappa

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species 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
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

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.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Blue Prestained Protein Marker, Broad Range (11-250 kDa): (#59329).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-mouse IgG, HRP-linked Antibody (#7076 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 262

Specificity / Sensitivity

MERS-CoV Spike Protein (E9P2L) Mouse mAb recognizes endogenous levels of total MERS-CoV spike protein. The antibody detects full-length MERS-CoV spike protein and also detects the S1 fragment generated by host protease cleavage. It does not cross-react with spike proteins from SARS coronaviruses (CoV-1, CoV-2).

Species Reactivity:

Virus

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val534 of MERS-CoV spike protein.

Background

Middle East Respiratory Syndrome (MERS) is a contagious viral respiratory infection, whose causative agent was identified as the Middle East Respiratory Syndrome-related coronavirus (MERS-CoV) (1,2). MERS-CoV is a single-stranded mRNA betacoronavirus; similar to other infectious coronaviruses (e.g., SARS-CoV-2), the MERS-CoV virion is comprised of four key structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) (3). Coronavirus spike proteins are class I fusion proteins that harbor an ectodomain, a transmembrane domain, and an intracellular tail. 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 (4). MERS-CoV spike proteins utilize the cell surface protein DPP4/CD26 as the receptor for cellular entry (5,6); notably, this protein displays no structural similarities to ACE2, the primary receptor for SARS-CoV-1 and SARS-CoV-2. 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 (7,8).
  1. Bermingham, A. et al. (2012) Euro Surveill 17, 20290.
  2. de Groot, R.J. et al. (2013) J Virol 87, 7790-2.
  3. van Boheemen, S. et al. (2012) mBio 3, e00473-12. doi: 10.1128/mBio.00473-12.
  4. Jiang, S. et al. (2013) J Infect 66, 464-6.
  5. Wang, N. et al. (2013) Cell Res 23, 986-93.
  6. Raj, V.S. et al. (2013) Nature 495, 251-4.
  7. Du, L. et al. (2009) Nat Rev Microbiol 7, 226-36.
  8. Yuan, Y. et al. (2017) Nat Commun 8, 15092.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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