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62405
Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate)
Antibody Conjugates
Monoclonal Antibody
R
Recombinant

Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) #62405

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Flow cytometric analysis of live pan-CD3+ T cells isolated from human PBMCs and engineered to express an scFv-based Anti-CD19 CAR containing a Whitlow/218 linker, using Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left). Tag Blue fluorescent protein (TagBFP) is co-expressed with the CAR. Data courtesy of Michael Kvorjak, Lohmueller lab (University of Pittsburgh).
Flow cytometric analysis of live pan-CD3+ T cells isolated from human PBMCs and engineered to express an scFv-based Anti-CD20 CAR containing a Whitlow/218 linker, using Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left). Tag Blue fluorescent protein (TagBFP) is co-expressed with the CAR. Data courtesy of Michael Kvorjak, Lohmueller lab (University of Pittsburgh).
Flow cytometric analysis of a mixed population containing live wild-type Jurkat cells and Jurkat cells engineered to express an scFv-based Anti-CD19 CAR containing a Whitlow/218 linker, using Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left). Tag Blue fluorescent protein (TagBFP) is co-expressed with the CAR. Cell line was provided by the Lohmueller Lab, University of Pittsburgh.
Flow cytometric analysis of live Jurkat cells (blue, negative) or Jurkat cells engineered to stably express an scFv-based Anti-CD19 CAR containing a Whitlow/218 linker (green, positive) using Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines). Cell line was provided by the Lohmueller Lab, University of Pittsburgh.
To Purchase # 62405
Cat. # Size Qty. Price
62405S
100 µl  (50 tests)
62405L
300 µl  (150 tests)

Supporting Data

REACTIVITY All
SENSITIVITY Transfected Only
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

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
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) fluorescent dye under optimal conditions and tested in-house for direct flow cytometric analysis in human cells. This antibody conjugate is expected to exhibit the same reactivity as the unconjugated Whitlow/218 Linker (E3U7Q) Rabbit mAb #57710, which is expected to react with cell surface expressed CARs of varying specificity containing a Whitlow/218 linker within the scFv of the extracellular domain.

Product Usage Information

Application Dilution
Flow Cytometry (Live) 1:50

Storage

Supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Live Cell Protocol for Directly Conjugated 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.

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: Human Fc receptors cross-react with rabbit IgG. When cells of interest express high levels of Fc receptor protein (for example, macrophage/monocyte lineages), pre-incubate live cells with human Fc block prior to immunostaining with rabbit antibodies.

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. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of Antibody Dilution Buffer and analyze on flow cytometer.

posted June 2017

revised January 2022

Protocol Id: 1504

Specificity / Sensitivity

Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) recognizes exogenously expressed levels of scFv-based CARs containing a Whitlow/218 linker.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues of the Whitlow/218 linker peptide.

Background

The Whitlow/218 peptide is a commonly used linker that connects the variable heavy (VH) domain and variable light (VL) domain of single-chain variable fragments (scFvs) and chimeric antigen receptors (CARs) that utilize an extracellular domain scFv for target antigen recognition. The Whitlow/218 linker affords the scFv with enhanced resistance to proteolysis and aggregation (1).

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.

For Research Use Only. Not for Use in Diagnostic Procedures.
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