Immunology methods 39

2.3.1 Generation of antibodies

2.3.1.1 Generation of polyclonal antisera against Pam17 protein

Polyclonal antisera were generated in rabbits. Recombinant protein was expressed in bacteria, purified using a specific tag and used as antigens. After the purification on the column remaining contaminants were separated from the proteins of interest by SDS- PAGE. Upon Western blotting, the bands corresponding to the protein of interest were excised from nitrocellulose membranes. Up to 200 µg of proteins (10 bands) were dissolved in 300 µl DMSO by vortexing for 3 min. TiterMax adjuvant (300 µl) was added and the emulsion injected subcutaneously into rabbits. The antigen was injected twice within ten days before the first bleeding was taken. All subsequent injections took place every four weeks. Freunds incomplete adjuvant was used instead of TiterMax adjuvant for all the injections except the first one. The rabbits were bled 10-12 days after each injection cycle. Approximately 30-40 ml of blood was taken from the ear vein and left to coagulate at RT for 2 h. Coagulated blood was centrifuged twice (5 min at 3000 x g and 15 min at 20000 x g, RT), and the supernatant was incubated at 56ºC for 20 min to inactivate complement system. The antisera prepared this way were then aliquoted and frozen at -20ºC.

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2.3.1.2 Affinity purification of antibodies against different components of the TIM23 complex

Affinity purification was performed in order to reduce the cross-reactivity of the antisera. Antibodies against Tim23, Tim44, Tim50, Tim16, Tim14, Tim17, Tim21 and Pam17 were purified on the affinity columns made by coupling the proteins that served as antigens to the CNBr-activated Sepharose 4B (Amersham) via their -NH2 groups. To remove all other amino group containing substances from protein in the solution prior to coupling, the buffer was exchanged with a bicarbonate one on the PD-10 column (Amersham). The column was equilibrated with 30 ml 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3 and 2.5 ml of solution containing 4-8 mg protein was loaded on the column by the gravity flow. First 2.5 ml of the eluate was discarded and the protein was collected from the column in the following 3.5 ml. During equilibration of the PD-10 column, CNBr- Sepharose was prepared in a way that 0.4 g of the beads was placed in 5 ml 1 mM HCl to swell. After 45 min the beads gave rise to ca. 1.5 ml gel. Gel was washed on a sintered glass filter with 200 ml 1 mM HCl and transferred into a column (max. volume 10 ml). Remaining HCl solution was allowed to pass through and the column was closed at the bottom. Upon addition of 3.5 ml of protein solution column was closed at the top and gently mixed by slowly revolving around vertical axis for 1 h at RT. The column was put in the vertical position; buffer was allowed to pass through and it was quickly analyzed for protein content with Ponceau S staining to check the efficiency of coupling. Remaining active groups were blocked by loading 6 ml 0.1 M ethanolamine, pH 8.0; 2 ml were allowed to pass through before the column was closed and gently mixed by slow revolving for additional 2 h at RT. Subsequently, the column was put in the vertical position; ethanolamine was allowed to pass through and all nonspecifically bound proteins were removed by 3 washing cycles of alternating pH. Each cycle consisted of 6 ml 0.1 M Na-acetate, 0.5 M NaCl, pH 4.5 followed by 6 ml 0.1 M Tris·HCl, 0.5 M NaCl, pH 8.0. Column was finally washed with 10 ml 10 mM Tris·HCl, pH 7.5, and it was ready for affinity purification of antibodies. If the antibodies were not purified the same day, 3 ml 0.05% NaN3 water solution was added and the column was stored at 4°C. Before purification, the column was left at RT for 45 min and then equilibrated with 10 ml of 10 mM Tris·HCl, pH 7.5. Antiserum (6 ml) was diluted with 24 ml 10 mM

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Tris·HCl, pH 7.5 and loaded on the corresponding affinity column under gravity flow. The column was washed with 10 ml 10 mM Tris·HCl, pH 7.5 followed by 10 ml 10 mM Tris·HCl, 0.5 M NaCl, pH 7.5. For the elution, column was subjected to alternating pH through application of 10 ml of each of the following buffers in given order: 10 mM Na- citrate, pH 4.0, 100 mM glycine·HCl, pH 2.5 and 100 mM Na2HPO4, pH 11.5. Fractions of 1 ml were collected and neutralized immediately with 200 µl 1 M Tris·HCl, pH 8.8 in the case of the first two buffers, and with 100 µl glycine, pH 2.2 in the case of the phosphate one. Several fractions eluted with each of the elution buffers were checked for specificity by immunodecoration on nitrocellulose membrane carrying yeast mitochondrial proteins. The majority of the specific antibodies were eluted with the glycine buffer in fractions 2-6. These fractions were usually pooled and 150 µl aliquots were stored at –20°C.

2.3.2 Immunodecoration

Proteins blotted onto nitrocellulose or PVDF membranes were visualized by immunodecoration with specific antibodies. After Western blot, membranes were incubated for 30 min in 5% (w/v) milk powder in TBS (150 mM NaCl, 10 mM Tris·HCl, pH 7.5) to block all nonspecific binding sites. The membranes were then incubated with specific primary antibody (1:100 to 1:20000 dilutions in 5% milk in TBS) for 1-2.5 h at RT, or overnight at 4ºC. The membranes were then washed for 5 min in TBS, 10 min in TBS containing 0.05% Triton X-100 and again 5 min in TBS and subsequently incubated with goat Anti-rabbit antibodies coupled to horseradish peroxidase (diluted 1:10.000 in 5% milk in TBS) for 1-2 h at RT. The membrane was then washed as already described, treated with the chemiluminescent substrate of peroxidase (ECL reagents 1 and 2) and the signals were detected on X-ray films (Fuji New RX).

ECL reagent 1: 3 ml Tris·HCl, pH 8.5 (1M stock), 300 µl luminol (440 mg/10 ml DMSO), 133 µl p-coumaric acid (150 mg/10 ml DMSO), H2O to 30ml.

ECL reagent 2: 3 ml Tris·HCl, pH 8.5 (1M stock), 18 µl H2O2 (30%), H2O to 30ml.

Solutions are stable for 7-10 days if kept in light-protected bottles at 4°C. Chemiluminescent substrate of peroxidase was made by mixing equal volumes of ECL reagents 1 and 2.

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