Protocol: Immunoprecipitation for MALDI mass spectrometry 

Carolyn Livsey

IP Buffer: (this is 1x but I usually make 10x stock)

0.1% Triton X-100
140 mM NaCl
0.025% Na Azide
10 mM Tris-HCl
protease inhibitors (Calbiochem cocktail)

(This protocol should be done in the cold room)
Step 1: to get rid of non-specific Ig binding to beads

add equal volumes of your sample and normal rabbit serum
1x IP buffer
rotate overnight
add beads (Calbiochem Protein G Plus / Protein A Agarose)
rotate 3 hours
centrifuge
save supernatant, discard beads

Step 2: immunoprecipitate

add antibody to supernatant
rotate overnight
add beads
rotate 3 hours
centrifuge
save beads, discard supernatant

Step 3: wash beads

add 500 µl IP buffer, rotate 10 minutes, centrifuge, discard supernatant
repeat 2 times with IP buffer
repeat 2 times with Tris HCL pH 8 (to get rid of protease inhibitors, salt, detergent)

Step 4: elute
elution buffer: TFA/H2O/acetonitrile 1 : 20 : 20

add 40 µl elution buffer to beads
rotate 10 minutes
centrifuge
save supernatant, discard beads

After elution, the sample can be analyzed by mass spectrometry to determine the molecular weight. I use Maldi-TOF (matrix-assisted laser desorption/ionization - time of flight).

To confirm a peptide sequence within a known protein, the peptide can be oxidized with 10µM H₂O₂. This oxidizes every methionine in the sample and shifts the molecular weight up by 16 for each methionine (R. Wang et al., 1996)

Wang R; Sweeney D; Gandy SE; Sisodia SS.
The profile of soluble amyloid beta protein in cultured cell media. Detection and quantification of amyloid beta protein and variants by immunoprecipitation-mass spectrometry.
Journal of Biological Chemistry, 1996 Dec 13, 271(50):31894-902