EphB2-Fc precipitates from rat hippocampal neurons in culture and from brain lysates
Lab/Group: Acker-Palmer Lab
Related Journal & Article Information
Journal: Nature Neuroscience
Article Title: Grb4 and GIT1 transduce ephrinB reverse signals modulating spine morphogenesis and synapse formation
Introduction
EphrinBs are transmembrane ligands for the Eph receptor tyrosine kinases. Here we describe the procedure used to precipitate ephrinB signaling complexes from hippocampal neurons in culture or brain lysates using soluble EphB receptor ectodomains fused to Fc portions of human IgG (EphB2-Fc).
Materials
Reagents
5 mg of mouse brain
106 rat hippocampal neurons in culture for 17 days in vitro
EphB2-Fc (200 μg/ml, R&D), Fc (1.8 μg/μl), anti-human Fc (μg/μl)
protein-G sepharose beads (GE)
LBA lysis buffer (50 mM Tris-HCl [pH 7.5], 0.5% Triton-X100, 150 mM NaCl, 10 mM NaPPi, 20 mM NaF, 1 mM sodium orthovanadate, 1% Complete (Roche))
PLC buffer (50 mM HEPES [pH 7.5]; 150 mM NaCl; 10% glycerol; 1% NP-40; 0.1% Triton-X100; 1.5 mM MgCl2; 1mM EGTA; 10mM NaP2O7; 100mM NaF; 1mM Na3VO4; 1% Complete (Roche))
HNTG buffer (20 mM HEPES [pH 7.5]; 150 mM NaCl; 10% glycerol; 0.1% Triton-X100; 1mM Na3VO4; 1% Complete (Roche))
Loading buffer for proteins
standard cell culture material and media
standard western-blotting material
Equipment
Standard cell culture equipment
Standared western blotting equipment
Procedure
A: Mouse brain
1. Homogenize a murine brain in 2 ml LBA lysis buffer
2. Spin down at 16,000 g, 10 min, 4oC
3. Take supernatans and measure protein concentration.
4. Incubate 1.5 mg protein with protein-G sepharose beads for 30 min at 4oC.
5. Spin samples down at 6,000 g, 5 min, 4oC.
6. Incubate overnight at 4oC these pre-cleared supernatans with protein-G Sepharose beads and 5 μg EphB2-Fc (precipitate) or Fc (control for unspecific protein binding).
7. Spin them down at 6,000 g, 5 min, 4oC.
8. Wash beads three times with HNTG buffer
9. Add 20 μl Loading buffer
10. Boil samples for 5 min. Load in a 9% SDS-PAGE gel and develop by Western blot with the desired antibodies.
B: Hippocampal neurons
B1. Stimulation of hippocampal neurons.
1. Cluster EphB2- Fc or Fc one hour prior stimulation at RT: Mix EphB2-Fc/Fc 1 μg/ml + 1/10 anti-hFc (0.1 μg/ml)
2. Wash neurons 2 x with HBSS.
3. Add EphB2-Fc/Fc clusters to pre-warmed NB medium.
4. Apply to the neurons and incubate at 37oC for 1 hr
B2. Neuron lysates
1. Place plates on ice and wash twice with cold PBS.
2. Lyse cells in PLC buffer; incubate at 4oC for 10 min; spin down samples at 16,000 g 10 min 4oC.
3. Take supernatans and measure protein concentration.
B3. EphB2-Fc precipitation
1. Incubate 3 mg total protein from the neuron lysates with protein-G Sepharose beads for 1 hr at 4oC.
2. Add 10 μg of EphB2-Fc or Fc to the appropriate sample
3. Incubate overnight at 4oC.
4. Spin them down at 6,000 g, 5 min, 4oC.
5. Wash beads three times with HNTG buffer
6. Add 20 μl Loading buffer
7. Boil samples for 5 min. Resolve in a 7.5% SDS-PAGE gel and develop by Western blot with the desired antibodies.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
EphB-Fc precipitates, ephrinB ligands, signaling complexes