Bulk purification of molecules of the major histocompatibility complex

Since the recent insight into the molecular basis of peptide presentation by molecules of the major histocompatibility complex (MHC), the bulk purification of peptide-MHC complexes has become established as a standard technique. It is now widely used in order to identify natural peptide ligands or peptide-binding characteristics of particular MHC specificities. In principle, this purification method can be considered as a large-scale immunoprecipitation procedure, which can be adapted for the bulk purification of any cell surface molecule.

The isolation of MHC-bound peptides

The MHC-peptide complexes are purified from cells of which pellets are collected at -70°C. A lysate (of 1010 cells or more) is made by thawing and resus-pending the cell pellet at a concentration of 2 X 10s cells ml1 in NP40-containing lysis buffer (described above). The lysate is precleared by incubation with Sepharose CL-4B beads (10 ml beads per 50 ml lysate; Pharmacia). This incubation can be done batch-wise in 50 ml tubes under quiet agitation for 2 h at room temperature. After the incubation the Sepharose beads are removed from the lysate using a glass filter that is placed over a vacuum. The prc-cleared lysate is subsequently incubated batch-wise for 16 h at 4°C with CNBr-activated Sepharose CL-4B beads to which antibody specific for the MHC molecule of interest has been covalently coupled (according to manufacturer's guidelines; Pharmacia). Then the affinity beads are washed on a glass filter (placed over a vacuum), taking care not to disturb the homogenous gel bed on the filter or letting the bed run dry. First, the beads are washed with minimally 5 bed volumes of a buffer containing 50 mM TRIS, 150 mM NaCl, pH 8.0, to which 0.5% NP40 (v/v; HPLC grade) has been added and subsequently with minimally 10 bed volumes of the TRIS/NaCl buffer without NP40. Then washing proceeds with 10 bed volumes 10 mM TRIS buffer, pH 8.0, and the beads are transferred and packed into a disposable column. The MHC-peptide complexes are eluted from the affinity beads with 3 bed volumes 10% acetic acid or 0.1% trifluoroacetic acid. The high molecular weight chains of the MHC molecules are removed from the eluate by ultracentrifugation over a membrane filter with a 10 kDa cut-off (Centriprep 10, Amicon). The ultimate peptide sample is concentrated by evaporation under vacuum and is ready for further processing.

The isolation of MHC molecules

In order to isolate intact MHC molecules for the purpose of (peptide) binding analyses, the procedure to follow is identical to that described for the isolation of MHC-bound peptides, except for the use of different buffers in the washing procedure and the elution of MHC under basic rather than acidic conditions. Briefly, the affinity beads that have been incubated with the precleared lysate are washed on a glass filter using the following buffers: minimally 5 bed volumes of TRIS/NaCl buffer (see above) with 0.5% NP40, 10 bed volumes of TRIS/NaCl buffer without NP40 and minimally 10 bed volumes of TRIS/NaCl with 0.4% (w/v) «-octylglucoside. In the latter buffer the beads are transferred to a disposable column and the

MHC complexes are eluted with 3 bed volumes of 50 mm diethylamine, 150 mm NaCl, 0.4% (w/v) n-octylglucoside, pH 11.5. The eluate can be neutralized using a 2m glycine solution (pH 2.5) and the high molecular weight MHC complexes in the eluate can be retained in a small volume after ultracentri-fugation over a membrane filter (10 kDa cut-off).

See also: Affinity chromatography; Antibodies, specificity; Isoelectric focusing; Radioiabeling; SDS-poly-acrylamide gel electrophoresis (SDS-PAGE).

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