Contact Us
800.227.0627

Recommended Glycine-based Immuno-precipitation procedure

 
Print Print
02/10/2011

Recommended Glycine-based Immuno-precipitation procedure

Preparation of Pre-Clearing beads
  • Bind 30 ml of pre-immune sera (or non-immune sera if pre-immune is not available) to 150ml of Protein A Sepharose beads for 2 hours at room temperature in 800 ml PBS
  • Wash beads 5 times with 1 ml of buffer A (250mM KCl, 0.1% IGEPAL CA-630)
Preparation of Immuno-Precipitation Beads
  • 50ml of IP beads were washed as shown below;
    • Buffer A + 250mM KCl, 0.1% (v/v) IGEPAL CA-630
    • Buffer A + 500mM KCl, 0.1% (v/v) IGEPAL CA-630
    • 10mM Phosphate buffer pH 6.8 o 2x 200mM Glycine pH 2.5
    • Buffer A + 250mM KCl, 0.1%(v/v) IGEPAL CA-630
    •  
Incubation of IP beads with Pre-Cleaned samples
  • Add 800uL of pre-cleared protein sample to the IP beads and incubate overnight at 4°C
  • Spin at 4K x g for 20 sec and collect non-bound supernatant
  • Spin supernatant at 13K x g for 1 min to remove Sepharose
Washing of IP beads and elution of proteins (All centrifugations to be carried out at 13K x g for 1 min)
  • Wash IP beads 2x in Buffer A + 250mM KCl, 0.1% (v/v) IGEPAL CA-630
  • Wash IP beads 3x in Buffer A + 500mM KCl, 0.1% (v/v) IGEPAL CA-630
  • Transfer to 0.5ml Eppendorf with last wash
  • Wash IP beads with 10mM Phosphate buffer pH 6.8
  • Elute proteins with 60 ml 200mM Glycine pH 2.5
    • Mix for 30 sec, centrifuge for 30 sec, mix for 30sec, centrifuge for 30sec
  • Neutralise by addition of 6 ml of 1.5M Tris. HCl pH 8.8.
  • Add 14 ml of Buffer A + 250mM KCl, 0.1% (v/v) IGEPAL CA-630 (Total volume 80 ml)
  • Spin at 13K x g for 1 min
Acetone Precipitation of Eluted Proteins
Add 4 volumes of ice-cold acetone, mix immediately and incubate at -20c overnight. Spin at max speed in an eppendorf centrifuge for 30 min, carefully remove supernatant and gently wash x1 with 90% methanol. Spin and air-dry (until just dry –do not over dry)