Immunoprecipitation via Biotinylation

Contents:

Prepare cells preparation, by washing 3 times in PBS (centrifuge at ª1200 rpm 4oC). Resuspend cells at 10^8/ ml in PBS.

Biotinylation Of Cell Surface Proteins

  • To 1 ml of cell suspension (10^8) add 50ul of fresh 0.1M NHSS-biotin in PBS (final conc n 5 mM biotin)
  • Incubate for 30 min at 4oC (occasional gentle mix)
  • Wash 3 times in PBS and resuspend at 2x10^7/ml in PBS.

Solubilization of Cells

  • To 1 ml of cells add 1 ml of Nonidet P40 buffer & 10ul of a protease inhibitor cocktail (see below).
  • Gently mix suspension and incubate for 30 min at 4oC.
  • The lysed cells are then centrifuged at 200g for 10 min at 4oC (removes nuclei and cell debri), keep supernatant.
  • Centrifuge supernatant at 100 000g for 30 min at 4oC

Preparation of protein G-Sepharose

  • Swollen protein G-Sepharose is washed in PBS, equilibrated and adjusted to 50% v/v with NP40 buffer. Use immediately for absorption of Ag-Ab complexes involving IgG 2a and IgG 2b subclasses.

Immunoprecipitation

  • To each 1 ml of NP40 lysate (in 1.5 ml Eppendorf) add 400ul of hybridoma culture supernatant.
  • Incubate at 4oC for 16 hours on rotating mixer.
  • Transfer to a new tube containing 25ul of 50% protein A-Sepharose (for IgG 2a and IgG 2b subclasses).
  • Incubate for 1 hour at 4oC on rotating mixer, and centrifuge for 10 sec in microfuge.
  • Quickly wash pellets 4 times (twice with NP40 buffer, once with 0.1% NP40, 50mM Tris-HCl, 150mM NaCl pH 8.0, once with 50mM Tris-HCl, 150mM NaCl, pH 8.0)
  • Store as pellet at -20oC prior to electrophoresis.

NHSS-biotin : sulfo-N-hydroxysuccinimide ester derivative of biotin.

Nonidet P40 buffer : 1% NP40, 1 mM EDTA, 50mM Tris-HCl, 150mM NaCl pH 8.0

Protease inhibitors: 0.0174g of phenylmethyl sulphonyl flouride(PMSF) made up to 1 ml in Iso-propanol (added at 5ul/ml of cells).

Aprotinin 5ul/ml of cells

Iodoacetamide 0.0925g in 1 ml of PBS (added at 5ul/ml of cells).

Western Transfer

  • After electrophoresis, transfer proteins onto nitrocellulose (50 Volts 40 min, then 75 Volts for 25 min at room temp).

  • Once transfer of proteins has occurred, block the nitrocellulose using either 3% Bovine Serum Albumin (BSA) in PBS 0.1% sodium azide or 3% skim milk powder 0.1% sodium azide in water. Block for 1 hr at 20oC.

Visualisation of Biotin

  • Wash 4 times in PBS-0.1% Tween 20 and once in PBS alone (5 min wash).
  • Add streptavidin biotinylated horseradish peroxidase (strep-bioHRP, Amersham ) diluted 1/400 in PBS-1.0% BSA. Incubate for 30 min at 20oC.
  • Wash 4 times in PBS-0.1% Tween 20 and once in PBS alone (5 min wash).
  • Add Western Blot Chemiluminescence reagents (DuPont Renaissance Western Blot Chemiluminescence Reagent. Cat. N o. NEL-100) to nitrocellulose for 1 minute then pour off.
    • (Add 2 ml of Bottle 1 :Enhanced Lumniol Reagent to 2 ml of Bottle 2 : Oxidizing Reagent (DO NOT CROSS CONTAMINATE). Apply to nitrocellulose).
  • Seal nitrocellulose in plastic (minus air bubbles) and place out of light.
  • Expose nitrocellulose to X-ray film and examine results.