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52342
SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Primary Antibodies
Monoclonal Antibody

SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb #52342

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  2. IF
Immunohistochemistry Image 1: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2 positive human placenta using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb.
Immunohistochemistry Image 2: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2 positive human placenta (top) or SARS-CoV-2 positive human lung (bottom) using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (left) compared to SARS-CoV-2 Nucleocapsid Protein (E8R1L) Mouse mAb #33717 (right). The differential signal observed between SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb and SARS-CoV-2 Nucleocapsid Protein (E8R1L) Mouse mAb #33717 antibodies is likely related to differences in antigen abundance of spike and nucleocapsid proteins.
Immunohistochemistry Image 3: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Immunohistochemical analysis of paraffin-embedded SARS-CoV-2-infected hACE2 K18 mouse lung using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (left) compared to secondary-only negative control (right). Note the mouse on mouse background staining in the absence of primary antibody. Human ACE2 transgenic mouse lung tissue was generously provided by Dr. Nicholas Crossland and Dr. Florian Douam, National Emerging Infectious Diseases Laboratories (NEIDL), Boston University.
Immunohistochemistry Image 4: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
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), squamous cell lung carcinoma (right-bottom), and ductal breast carcinoma (left-bottom) using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse 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 5: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Immunohistochemical analysis of paraffin-embedded 293T cell pellet, untransfected (left) or SARS-CoV-2 spike protein transfected (right), using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb.
Immunofluorescence Image 1: SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb
Confocal immunofluorescent analysis of HCT 116 cells transiently transfected with SARS-CoV-2 spike protein (left, positive) or mock transfected (right, negative), using SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb (green), β-Actin (13E5) Rabbit mAb #4970 (red), and DAPI #4083 (blue).
To Purchase # 52342
Cat. # Size Qty. Price
52342S
100 µl $ 285

Supporting Data

REACTIVITY Vir
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2a

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
Immunohistochemistry (Paraffin) 1:200 - 1:800
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800

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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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Mouse #8125) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Mouse #8125) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

DETECTION REAGENT/SUBSTRATE COMPATIBILITY
RECOMMENDED
DETECTION REAGENTS
SignalStain® Boost IHC Detection Reagent (HRP, Mouse) #8125 SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926
COMPATIBLE
CHROMOGEN
SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632  

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.


posted February 2010

revised June 2020

Protocol Id: 280

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Methanol, 100%
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
  2. Allow cells to fix for 15 minutes at -20°C.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse in 1X PBS as in step 5.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  9. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 145

Specificity / Sensitivity

SARS-CoV-1/2 Spike Protein (2B3E5) Mouse mAb recognizes endogenous levels of total SARS-CoV-1 and SARS-CoV-2 spike protein.

Species Reactivity:

Virus

Source / Purification

Monoclonal antibody is produced by immunizing animals with SARS spike recombinant protein.

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.

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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