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58036
Protein L (PE Conjugate)
Antibody Conjugates
Miscellaneous

Protein L (PE Conjugate) #58036

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Flow cytometric analysis of live Jurkat cells (blue) or live Jurkat cells engineered to stably express an scFv-based Anti-CD20 CAR (green), using Protein L (PE Conjugate) (solid lines) or unstained cells (dashed lines). Cell line was provided by the Lohmueller Lab, University of Pittsburgh.
Flow cytometric analysis of live Jurkat cells (blue) or live Jurkat cells engineered to stably express an scFv-based Anti-CD19 CAR (green), using Protein L (PE Conjugate) (solid lines) or unstained cells (dashed lines). Cell line was provided by the Lohmueller Lab, University of Pittsburgh.

Product Usage Information

Recommended Dilution: 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. Protect from light. Do not freeze.

Protocol

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Flow Cytometry, Live Cell Protocol for Directly Conjugated Protein L

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. Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616) that is compatible with Protein L, 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: 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 Protein L conjugate, prepared in 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 Dilution Buffer. Discard supernatant. Repeat. If using a fluorescent conjugate of Protein L, skip to step 9.
  6. For labeling biotinylated Protein L, resuspend cells in 100 µl of diluted fluorochrome-conjugated streptavidin or anti-biotin antibody (prepared in Dilution Buffer at the recommended dilution).
  7. Incubate for 30 min on ice. Protect from light.
  8. Wash by centrifugation in Dilution Buffer. Discard supernatant. Repeat.
  9. Resuspend cells in 200-500 µl of Dilution Buffer and analyze on a flow cytometer.

Protocol Id: 2589

Product Description

This Cell Signaling Technology product is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells.

Specificity / Sensitivity

Protein L (PE Conjugate) is recombinantly derived and genetically engineered such as to remove the cell wall-, cell membrane-, and albumin-binding regions of Protein L to enhance binding to immunoglobulins. Conjugation to PE makes this product ideally suited for direct flow cytometric analysis of cells expressing κ light chain-containing immunoglobulins as well as immunoglobulin fragments that contain κ light chains, such as scFvs.

Species Reactivity:

Human, Mouse, Rat, Pig

Background

Protein L was first isolated from the surface of bacterial species Peptostreptococcus magnus (1). Unlike Protein A and Protein G that differentially bind to the Fc region of immunoglobulin isotype heavy chains, Protein L contains Ig binding domains that interact with the κ light chain variable domain without disrupting paratope function (2-4). This attribute not only facilitates binding to a broader range of immunoglobulin classes and subclasses than either Protein A or Protein G but also allows for binding to Fab fragments and single chain variable fragments (scFvs) that contain κ light chain sequences. Protein L does, however, display specificity with respect to κ light chain subtypes. In human, Protein L binds to VκI, VκIII, and VκIV subtypes whereas binding is restricted to VκI in mice (4).

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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To Purchase # 58036
Cat. # Size Qty. Price
58036S
100 µl  (50 tests)