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3419
Streptavidin (Sepharose® Bead Conjugate)
WB & IP Reagents
Miscellaneous

Streptavidin (Sepharose® Bead Conjugate) #3419

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  1. IP
Immunoprecipitation of extracts from COS cells transfected with Myc-Chorionic Somatomammotropin Hormone-Like 1 (CSL) protein using Myc-Tag (9B11) Mouse mAb (Biotinylated) #2084 and Immobilized Streptavidin (Bead Conjugate) (Lane 1) shows immunocomplexes pulled down using Streptavidin (Sepharose® Bead Conjugate). Lysate and beads alone are shown in lane 2 indicating the specificity of the streptavidin beads. Western blotting was performed using Myc-Tag (9B11) Mouse mAb (HRP Conjugate) #2040.
Immunoprecipitation of extracts from NIH/3T3 cells using PTEN (138G6) Rabbit mAb (Biotinylated) #9583 and Streptavidin (Sepharose® Bead Conjugate) (Lane 1). Lysate and beads alone are shown in lane 2 indicating the specificity of the streptavidin beads. Western blotting was performed using PTEN (26H9) Mouse mAb #9556.
Immunoprecipitation of extracts from Jurkat cells using Phospho-Akt (Thr308) (244F9) Rabbit mAb (Biotinylated) #3454 and Streptavidin (Sepharose® Bead Conjugate) (Lane 1). Lysate and beads alone are shown in lane 2 indicating the specificity of the streptavidin beads. Western blotting was performed using Phospho-Akt (Thr308) (L32A4) Mouse mAb #5106.

Product Usage Information

Add 10 μl of well-vortexed beads to 200 μl of cell lysate at 1 mg/ml pre-incubated with biotinylated primary antibody. See protocol for more details.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.

Protocol

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Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

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. Streptavidin (Sepharose® Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per immunoprecipitation.
  5. 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.
  6. 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)

  1. Add 10 µl Streptavidin (Sepharose® Bead Conjugate) (#3419; for biotinylated antibodies), to 200 µl cell lysate at 1 mg/ml.
  2. Incubate with rotation at 4°C for 30–60 min.
  3. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  4. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Gently mix Streptavidin (Sepharose® Bead Conjugate) (#3419) and add 10 µl of slurry. Incubate with rotation for 2 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
  4. Proceed to sample analysis 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 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: When using primary antibodies produced in rabbit to to detect proteins with a molecular weight in the range of 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) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse heavy or light chain.

When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.

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 (4–20%).

posted December 2008

revised October 2021

Protocol Id: 412

Product Description

Streptavidin (Sepharose® Bead Conjugate) is useful for the precipitation of biotinylated proteins (1,2). Recombinant streptavidin is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads.

Specificity / Sensitivity

Streptavidin has a remarkably high affinity for its natural ligand, biotin. The complex and irregular structure of the biotin-binding site makes it highly optimized for biotin binding and confers great specificity to the streptavidin-biotin complexes (4).

Source / Purification

Streptavidin is expressed in Escherichia coli.

Background

Streptavidin is a 53,000 dalton tetrameric protein purified from the bacterium Streptomyces avidinii (3). Each subunit binds to biotin with extremely high affinity. Because of its strong non-covalent interaction with biotin, streptavidin can be used to isolate biotinylated proteins (1,2).

Limited Uses

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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 # 3419
Cat. # Size Qty. Price Inventory
3419S
400 µl  (40 immunoprecipitations)