Competitive Ligand Binding to Cortical Receptor using Crossl

Sunday, October 19, 2003

Description
This protocol describes a competitive ligand binding assay for cortical neurotrophin receptors. Following binding in the presence of competitor, the bound radiolabeled ligand is cross-linked to the receptor. The cells are lysed and the ligand-receptor complexes are immunoprecipitated using a pan-trk (tyrosine kinase receptor) antibody.

Procedure
A. Preparation of Cortical Tissue for Competitive Crosslinking

1. Collect the cortices from adult rats (or mice) and immediately place in ice-cold PBS/Glucose Solution.

2. Replace the PBS/Glucose Solution several times to wash away any blood.

3. Remove two whole cortices and place in 10 ml of fresh ice-cold PBS in a 15 ml tube.

4. Gently homogenize using 20 ml syringe with an 18-gauge needle.

5. Allow the homogenate to sediment for 1 to 2 min on ice.

6. Collect the supernatant.

7. Centrifuge the supernatant at approximately 5,000 rpm for 5 min in a microcentrifuge and discard the supernatant.

8. Resuspend the pellet in 5 ml of ice-cold Binding Buffer.

B. Competitive Binding

1. Remove 1 ml aliquots of resuspended pellet and place in 1.5 ml microcentrifuge tubes.

2. Add different unlabeled Neurotrophin competitors (as determined by experimental design) at 50-fold excess to each tube. Set up one tube without any Neurotrophin as a negative control.

3. Add 1 nM of Iodinated Neurotrophin, cognate to the receptor under study, to each tube (see Radioligand Displacement Binding Assay for Neurotrophins protocol) (CAUTION! see Hint #1)

4. Incubate the samples on ice for two hr, resuspending the cells every 10-15 min.

5. Centrifuge the cells at 2,000 rpm for 5 min in a microcentrifuge at 4C and discard the supernatant.

6. Resuspend the pellet in 1 ml of ice-cold Binding Buffer.

7. Centrifuge the cells at 2,000 rpm for 5 min in a microcentrifuge at 4C and discard the supernatant.

8. Again, resuspend the pellet in 1 ml of ice-cold Binding Buffer.

C. Crosslinking Ligand to Receptor

1. Add cross-linker of choice to a final concentration of 2 mM and incubate for 30 min at room temperature, resuspending the cells every 10 min (see Hint #2).

2. Stop the cross-linking reaction by the addition of 1 M Tris, pH 7.5 to a final concentration of 20 mM.

3. Incubate for 10 min more.

4. Centrifuge the cells at 2,000 rpm for 5 min in a microcentrifuge and discard the supernatant.

5. To the cell pellet, add 1 ml of PBS, mix well, centrifuge at 2,000 rpm for 5 min in a microcentrifuge to pellet the cells and discard the supernatant.

6. Repeat the PBS wash in step 5.

D. Lysis and Immunoprecipitation

1. Resuspend the pellet in 500 l of Lysis Buffer and leave the tubes on ice for 10 min.

2. Vortex the samples briefly and then centrifuge for 5 min at 5,000 rpm in a microcentrifuge to remove debris.

3. Collect the supernatants and transfer into new microcentrifuge tubes.

4. Add 2 l of pan-trk antibody to each tube.

5. Incubate the samples for 3 hr at 4C.

6. Add approximately 70 l of Protein A Sepharose beads.

7. Incubate for 15 to 60 min at 4C on a cell shaker or rotator.

8. Centrifuge the beads at full speed for 1 to 2 min in a microcentrifuge and discard the supernatant.

9. Add 1 ml of Lysis Buffer, mix well and centrifuge the beads.

10. Discard the supernatant and repeat bead washing in lysis buffer.

E. SDS-PAGE Gel Eletrophoresis of Samples

1. Resuspend the beads in 40 l of 1X SDS-PAGE Sample Buffer.

2. Boil the samples for 5 min.

3. Fractionate proteins on a denaturing SDS-Polyacrylamide gel (see Denaturing SDS-PAGE Gel protocol).

4. Fix the gel in 30% Acetic Acid:Methanol Solution for 30 min at room temperature.

5. Incubate the gel in Amplify Solution for 15 min to amplify the radioisotope signal.

6. Dry the gel using a vacuum gel drier.

7. Expose the dried gel to X-ray film at -70C (exposure time must be determined empirically).

8. Develop the film and determine expression levels by densitometric scanning.

Recipes
PBS/Glucose Solution Prepared in PBS
5 mg/ml Glucose

PBS 1.7 mM Potassium Phosphate Monobasic (KH2PO4)
2.7 mM KCl
Adjust the pH to 7.4 using HCl
137 mM NaCl
10 mM Sodium Phosphate Dibasic (Na2HPO4)

Pan-tyrosine Kinase Receptor (trk) Antibody (see Hint #3)

Bifunctional, Membrane-impermeant Crosslinker such as DSS, DSP, BS3, EDAC (see Hint #2)

Acetic Acid:Methanol Solution 30% (v/v) Acetic Acid
10% (v/v) Methanol (CAUTION! see Hint #1)

SDS-Page Sample Buffer 7.5% Glycerol
5% 2-Mercaptoethanol (CAUTION! see Hint #1)
2% SDS
12.5 mM Tris-Cl, pH 6.8
0.01% Bromophenol Blue

Lysis Buffer 1 mM PMSF (CAUTION! see Hint #1)
1% (v/v) NP-40
1 mM Sodium Orthovanadate (Sigma) (CAUTION! see Hint #1)
20 mM Leupeptin (Sigma)
2 mM EDTA
0.15 unit/ml Aprotinin (Sigma)
10% (v/v) Glycerol
137 mM NaCl
20 mM Tris-HCl, pH 8.0

1 M Tris-Cl, pH 7.5

Binding Buffer 0.1 M Sodium Phosphate Buffer, pH 6.5
2% Bovine Serum Albumin (BSA)
0.7 M CaCl2

Supplies

Tips
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for

proper handling instructions.

2. A stock solution of DSS or DSP can be made in dry DMSO to a concentration of 10 to 25 mM. Prepare BS3 immediately before use. Make a stock solution in 5 mM Sodium citrate, pH 5.0. See manufacturer's instructions for further details.

3. Pan-tyrosine kinase receptor (trk) antibody from Santa Cruz Biotechnology has been used in this protocol.

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Competitive Ligand Binding to Cortical Receptor using Crossl