Tight Junction PROTOCOLS
Immunoprecipitation
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Tight junction complexes can be immunoprecipitated using antibodies against different tight junction proteins. The detergents of choice is CHAPS. Different TJ complexes can be isolated using ZO-1, ZO-2, occludin, and PKC zeta antibodies.
(1) Adherent epithelial cells are plated at 90-100% confluent density at 18 hours before utilization for biochemistry.

(2) On the first day of purification, cells are quickly rinsed four times with ice cold 10 mM HEPES, pH 9.0, and incubated in the same buffer for three hours at 4C without agitation

(3) At the end of three hours, in the cold room, the buffer is decanted quickly and gently to avoid sloughing off of cells from the plate.

(4) To maintain the highest protein concentration possible in the initial cell lysate, the remaining buffer is removed as much as possible by tilting the plate and using a pipetman.

(5) Cell are immediately scraped off the plates and collected in a 50 ml conical tube chilled on ice.

(6) After all the cells are collected from the plates, they are passed 10 times through a 22 G needle. Efficiency of homogenization is examined under phase-contrast of trypan blue stained whole cell lysate.

(7) After homogenization, protease inhibitors (Antipain, 50 μg/ml; Aprotinin, 2 μg/ml; calpain inhibitor I, 17 μg/ml; calpain inhibitor II, 7 μg/ml; E-64, 10 μg/ml; leupeptin, 1 μg/ml; Pefabloc SC, 1 mg/ml; pepstatin A, 1 μg/ml; Roche Molecular Biochemicals) are added immediately with continuous vortex.

(8) Large organelles, nuclei, and whole cells are removed by centrifugation at 30,000xg for 10 minutes at 4C (or 10,000xg for 30 min).

(9) The supernatant is centrifuged at 100,000 g for 60 minutes at 4C onto a saturated sucrose cushion.

(10) Membranes are collected at the buffer and sucrose interface, washed with ice-cold distilled water, and re-centrifuged at 100,000 g onto a saturated sucrose cushion in a new tube.

(11) The washed membranes are collected at the water/sucrose interface and diluted in 10% CHAPS in distilled water (chilled to 4C) to a final concentration of 2% CHAPS.

(12) The membranes are then incubated with primary antibodies (1-10 ug per 150 mm dish of cells dependent on the copy number per cell of the protein) for 18 hours at 4C.

(13) Membranes are centrifuged at 10,000 g for 30 min at 4C to remove precipitates.

(14) Cleared supernatant is incubated with Protein A-Sepharose beads (amount of beads needed depends on the binding capacity of the beads and the amount of primary antibodies added; in general, add two-fold excess of beads to pull down all antibody-antigen complexes). Rotate the beads for 18 hours at 4C.

(15) Beads are washed 6 times with ice cold 2% CHAPS in distilled water. Antibody-antigen complexes can be eluted with a peptide antigen in 100 mM NaCl, 15 mM HEPES, pH 7.5, for 60 min at 25C. Add at least a ten-fold molar excess of peptide should be added for the elution step. If a peptide antigen is not available, partial elution can be performed using high salt (0.5 M Tris, pH 7.5).

(16) A small fraction of the eluted immuno-complex can be used immediately for negative stain electron microscopy. The rest of the sample is passed through a 25G Sepharose spin column to remove salts and subsequently concentrated using a speedvac. The concentrated samples can be used immediately for gel analysis or stored at -80C.

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