This is a cached page for the URL (http://www.bdbiosciences.com/pharmingen/protocols/S_Labeled_Cells.shtml). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
BD Biosciences : Pharmingen : Protocols Becton Dickinson BD Biosciences Brands More Search Options Sitemap
BD Biosciences: Clontech, Discovery Labware, Immunocytometry Systems, Pharmingen
BD Biosciences > Pharmingen > Protocols >  Privacy | Terms & Conditions


Pharmingen

P r o t o c o l s

Immunoprecipitation of 35S Labeled Cells

 

  1. Cell Labeling
    1. Start with 1-5 x 106 cells that are in log phase growth
      1. For suspension cells, passage cells several hours prior to harvest for metabolic labeling. Cells that have recently been split may be in stationary phase and may not incorporate the radiolabel as efficiently as proliferating, log-phase cultures.
      2. For adherent cells, plate cells 1 day prior to metabolic labeling. Cells that are approximately 70-80% confluent are optimal, however this may vary according to cell type.
    2. Wash the cells in warm (37°C), serum-free medium (e.g., DMEM) which does NOT contain cysteine or methionine.
    3. Resuspend the cells in cysteine/methionine-free medium, containing 2% dialyzed fetal calf serum (FCS) and incubate at 37°C for 30 min (in the presence of CO2, if normally required).

      NOTE: Follow the radioisotope safety guidelines for all subsequent handling.

    4. Add 100 mCi/ml each of L-[35S]Cys and L-[35S]Met to the cells and mix well. Incubate the cells for 1-4 hr at 37°C, with occasional mixing to resuspend the cell pellet.
  2. Cell Lysis
    1. Wash the cells 3 times in ice cold PBS.
    2. Harvest the cells, gently scraping the plate if adherent cells are used. Centrifuge to pellet cells.
    3. Lyse cells by resuspending the cell pellet in 10 volumes of pre-chilled lysis buffer* or pre-chilled RIPA buffer* containing protease inhibitors*.
    4. Incubate on ice for 30 min.
    5. Centrifuge at 10,000 x g in a pre-chilled centrifuge/rotor (4°C) for 10-15 min.
    6. Collect the supernatant from the cell lysate and transfer to a new tube.

      *see recipe at end of protocol.

  3. Preclear Lysate
    1. Preclear the lysate by incubating with a control antibody (10 mg/ml for a monoclonal antibody; 1-5 ml for polyclonal antibodies) for 1 hr at 4°C with agitation. NOTE: Control antibody should be the same isotype as the primary monoclonal antibody; polyclonal antisera should be from the same animal (pre-immunization) or same species as the primary, specific antibody used.
    2. To 1 ml of cell lysate: Add 500 ml Protein G Sepharose® or Protein A Sepharose® bead slurry (10% v/v in lysis buffer or RIPA buffer containing protease inhibitors)
    3. Centrifuge at 10,000 x g for 15 sec at 4°C. Collect and aliquot the supernatant for precipitation with specific antibodies.
  4. Incubation with Specific Antibody
    1. Using approximately 50 mg of total cellular protein, (e.g., lysate from ~2 x 105 cells), add 1-5 mg of antibody per reaction.
    2. Incubate with rotation or agitation at 4°C for 2 hr.
  5. Immunoprecipitation
    1. Add an equal volume of Protein G Sepharose® or Protein A Sepharose® bead slurry to cell lysate. Incubate with rotation or agitation at 4°C for an additional 1-2 hr. NOTE: The choice of Sepharose® used is dependent on the isotype of the primary antibody to be used and the species in which it was made (see table 3)
    2. Centrifuge at 10,000 x g for 15 sec at 4°C.
    3. Aspirate the supernatant. Alternatively, the supernatant containing radiolabeled proteins may be transferred to a separate tube for immunoprecipitation with an additional/separate-specificity antibody.
  6. Wash
    1. Wash the beads 3-4 times in cold lysis buffer containing 0.5 M NaCl. Use RIPA Buffer for a more stringent wash.
    2. Transfer the beads to a fresh tube and wash once more with cold lysis buffer.
    3. After the last wash, add 50 ml of 1X SDS-PAGE sample buffer* to beads. Elute the bound immunocomplex by boiling the beads for 2 min.

    4. Centrifuge at 10,000 x g for 1 min before loading supernatant on gel.

      *see recipe at end of protocol

  7. Detection
    1. Resolve by SDS-PAGE.
    2. Stain gel with coomassie blue or silver stain, destain.
    3. Dry gel.
    4. Expose to X-ray film.
Protocols Index>>
Flow Cytometry
Fluorochrome Absorption and Emission Spectra
Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis
Cell Fixation/Permeabilization Kits
Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis
Annexin V Staining Protocol
APO-BRDU™ Procedure
APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Blockô in Immunophenotyping of Mouse or Rat Leukocytes
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunoprecipitation
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation




All trademarks are property of Becton, Dickinson and Company unless otherwise noted.
© Copyright 2003 Becton, Dickinson and Company


Contact Information | Privacy | Terms & Conditions | Sitemap