Order 3, Get Your 4th Free!* | Learn More >>
26902
Nectin-2/CD112 (E4H9V) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

Nectin-2/CD112 (E4H9V) Rabbit mAb #26902

Reviews ()
Citations (0)
Filter:
  1. IP
  2. F
Immunoprecipitation Image 1: Nectin-2/CD112 (E4H9V) Rabbit mAb

Immunoprecipitation of Nectin-2/CD112 protein from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Nectin-2/CD112 (E4H9V) Rabbit mAb. Western blot analysis was performed using Nectin-2/CD112 (E4H9V) Rabbit mAb.

Flow Cytometry Image 1: Nectin-2/CD112 (E4H9V) Rabbit mAb

Flow cytometric analysis of live MOLT-4 cells (blue, negative) and MCF7 cells (green, positive) using Nectin-2/CD112 (E4H9V) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

To Purchase # 26902S
Product # Size Price
26902S
100 µl $ 268

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 70-80
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • 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
  • 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
Immunoprecipitation 1:100
Flow Cytometry 1:100 - 1:400

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

PRINT

View >Collapse >

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.

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 of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 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.

  3. 3X 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.
  4. Protein A Magnetic Beads: (#73778).
  5. Magnetic Separation Rack: (#7017) or (#14654).
  6. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

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.

  1. Briefly vortex the stock tube to resuspend the magnetic beads.

    IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:

  2. Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

    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.

  3. Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

    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.

  4. Incubate with rotation for 20 minutes at room temperature.
  5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
  6. Proceed to immunoprecipitation section.

Immunoprecipitation

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

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex.
  2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
  3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
  4. Incubate with rotation for 20 min at room temperature.
  5. Pellet beads using magnetic separation rack. Wash pellets five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  6. Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
  2. Heat the sample to 95-100°C for 5 min.
  3. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

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).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15-30 µl) on SDS-PAGE gel.

posted December 2008

revised April 2018

Protocol Id: 410

Flow Cytometry, Live Cell Protocol for Unconjugated Rabbit Antibodies

A. Solutions and Reagents

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  3. Recommended Anti-Rabbit secondary antibodies::
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
    • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 30 min to 1 hr on ice. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min on ice. Protect from light.
  8. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

revised June 2020

Protocol Id: 1865

Specificity / Sensitivity

Nectin-2/CD112 (E4H9V) Rabbit mAb recognizes endogenous levels of total Nectin-2/CD112 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human Nectin-2/CD112 protein.

Background

Nectin-2, also known as CD112 and poliovirus receptor-related 2 (PVRL2), is a single-pass type I membrane glycoprotein ubiquitously expressed on various human tissues (1). It is a calcium independent cell adhesion molecule known to interact with several cell surface receptors, including DNAM-1 (CD226), LFA-1 (CD11a), Nectin-3 (CD113), TIGIT (VSTM3), and PVRIG (CD112R) (2-7). It is one of the major constituents of adherens junctions, and therefore plays a central role in a number of cellular processes, including adhesion, migration, and proliferation (2-8). Within the immune system, Nectin-2 modulates immune cell signaling. Upon interaction with DNAM-1 expressed on T and NK cells, Nectin-2 stimulates proliferation and cytokine production (4). Upon interaction with PVRIG, Nectin-2 inhibits proliferation (7). Thus, Nectin-2 can be either a co-stimulator or a co-inhibitor of immune cell function depending on competitive receptor interactions. Nectin-2 also serves as an entry for certain mutant strains of herpes simplex virus and pseudorabies virus, and it is involved in cell to cell spreading of these viruses (8,9). Alternate transcriptional splice variants, encoding different isoforms, have been characterized (10-12).

  1. Eberlé, F. et al. (1995) Gene 159, 267-72.
  2. Lopez, M. et al. (1998) Blood 92, 4602-11.
  3. Satoh-Horikawa, K. et al. (2000) J Biol Chem 275, 10291-9.
  4. Bottino, C. et al. (2003) J Exp Med 198, 557-67.
  5. Reymond, N. et al. (2004) J Exp Med 199, 1331-41.
  6. Deuss, F.A. et al. (2017) J Biol Chem 292, 11413-22.
  7. Zhu, Y. et al. (2016) J Exp Med 213, 167-76.
  8. Devilard, E. et al. (2013) PLoS One 8, e77424.
  9. Martinez, W.M. and Spear, P.G. (2001) J Virol 75, 11185-95.
  10. Warner, M.S. et al. (1998) Virology 246, 179-89.
  11. Ota, T. et al. (2004) Nat Genet 36, 40-5.
  12. Gerhard, D.S. et al. (2004) Genome Res 14, 2121-7.

Pathways & Proteins

Explore pathways + proteins related to this product.

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's 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's 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.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.