D:\106729882.doc
1
Immunoprecipitation with Soluble Antibodies
I.
1X IP Buffer (Crude Homogenates from Whole Heart)
Stock Solution
Final Concentration
Triton X-100
NaCl
Tris, pH 7.4
EDTA
EGTA
Sodium Vanadate
PMSF
NP-40
1%
150 mM
10mM
1 mM
1mM
0.2mM
0.2mM
0.5%
II.
Homogenization and Protein Measurement
1.
2.
3.
4.
Homogenize frozen tissue in 1:20 dilution (M/V) with buffer.
Shake on ice for 1 hour.
Centrifuge samples in microfuge for 30 minutes at 4°C. The supernatant is the
“total cell lysate (native)”.
Measure total protein concentration in supernatant.
III.
Immuprecipitation
1.
2.
Pretreatment…. (Ask DAVE)
Add 1-5ug of antibody, 500ul 2X IP buffer, and 100ul total lysate contaiing 200500ug total protein.
Vortex and incubate at 4°C for 1 hour. (If monoclonal Ab are used add 5ug rabbit
anti-mouse Ig antibody, vortex, and continue the incubation for an additional 30
minutes at 4°C)
Add 10ul 50% Protein A-Agarose.
Vortex and incubate with agitation for 30 minutes at 4°C.
Wash 3 times by centrifugation resuspension with 1X IP buffer, followed by
collection by centrifugation for 4 minutes in a microcentrifuge.
Resuspend pellet in 30ul 2X concentrated electrophoresis sample buffer
Boil for 5 minutes and centrifuge for 5 minutes.
Load supernatant onto an SDS-PAGE gel.
3.
4.
5.
6.
7.
8.
9.
Last Updated: January 14, 1999 3:14 PM