EphB2-Fc precipitates from rat hippocampal neurons in culture and from brain lysates

doi:10.1038/nprot.2007.163

Method Article

EphB2-Fc precipitates from rat hippocampal neurons in culture and from brain lysates

https://doi.org/10.1038/nprot.2007.163

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Biological techniques

Neuroscience

EphB-Fc precipitates

ephrinB ligands

signaling complexes

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).

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

Standard cell culture equipment

Standared western blotting equipment

A: Mouse brain

Homogenize a murine brain in 2 ml LBA lysis buffer
Spin down at 16,000 g, 10 min, 4^oC
Take supernatans and measure protein concentration.
Incubate 1.5 mg protein with protein-G sepharose beads for 30 min at 4^oC.
Spin samples down at 6,000 g, 5 min, 4^oC.
Incubate overnight at 4^oC these pre-cleared supernatans with protein-G Sepharose beads and 5 μg EphB2-Fc (precipitate) or Fc (control for unspecific protein binding).
Spin them down at 6,000 g, 5 min, 4^oC.
Wash beads three times with HNTG buffer
Add 20 μl Loading buffer
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.

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)
Wash neurons 2 x with HBSS.
Add EphB2-Fc/Fc clusters to pre-warmed NB medium.
Apply to the neurons and incubate at 37^oC for 1 hr

B2. Neuron lysates

Place plates on ice and wash twice with cold PBS.
Lyse cells in PLC buffer; incubate at 4^oC for 10 min; spin down samples at 16,000 g 10 min 4^oC.
Take supernatans and measure protein concentration.

B3. EphB2-Fc precipitation

Incubate 3 mg total protein from the neuron lysates with protein-G Sepharose beads for 1 hr at 4^oC.
Add 10 μg of EphB2-Fc or Fc to the appropriate sample
Incubate overnight at 4^oC.
Spin them down at 6,000 g, 5 min, 4^oC.
Wash beads three times with HNTG buffer
Add 20 μl Loading buffer
Boil samples for 5 min. Resolve in a 7.5% SDS-PAGE gel and develop by Western blot with the desired antibodies.

Hippocampal neuron cultures: 17 days

Precipitation: 2 days

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EphB2-Fc precipitates from rat hippocampal neurons in culture and from brain lysates

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EphB2-Fc precipitates from rat hippocampal neurons in culture and from brain lysates

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Reagents

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Associated Publications

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Terms of Service

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