42. Combs.
43. Coomassie Brilliant Blue: 2.5 mg/mL solution. Weigh out
0.25 g Coomassie Brilliant Blue, dissolve in 10 mL of glacial
acetic acid, and add 1/1 (vol/vol) methanol and water to a
final volume of 100 mL. Stir the solution for 3–4 h and then
filter through filter paper. Store at room temperature.
44. Destaining solution: 10% acetic acid, 45% methanol. Mix
10 mL of glacial acetic acid and 45 mL of methanol and add
water to a final volume of 100 mL.
45. Dialysis membrane with a cutoff of 10 kDa.
2.3 Interaction Assay
1. ELISA microplates (96-well) for high binding.
2. Absorbance microplate reader.
3. 37
C incubator.
4. Microplate washer.
5. 50 mL tubes.
6. 1.5 mL vial.
7. Sealing tape for 96-well plates.
8. Wash buffer: 1Â PBS, 0.05% Tween 20 (PBS-T).
9. Blocking buffer: PBS-T, 10% dry milk or 1% BSA. Weigh out
10 g skimmed dry milk, and dilute the powder in 100 mL
PBS-T. For preparing BSA, weigh out 1 g BSA, and dissolve
in 100 mL of PBS-T.
10. Mouse antiserum against protein of interest.
11. HRP-conjugated anti-His monoclonal antibodies.
12. HRP-conjugated anti-mouse IgG.
13. Bovine serum albumin: 1 mg/mL in water. Aliquot and store
at À20
C.
14. Fetuin from fetal bovine serum: 1 mg/mL in water. Aliquot
and store at À20
C.
15. o-Phenylenediamine (OPD).
16. Hydrogen peroxide (H 2 O 2 ).
17. Citrate-phosphate buffer: 0.1 M citrate, 0.2 M phosphate,
pH 5.0.
18. Sulfuric acid: 2 M solution in water.
3 Methods
3.1 Cloning
and Expression
of Recombinant
Protein in E. coli
1. Genes of interest are amplified without the signal peptide (see
Note 7) from the genomic DNA of L. interrogans by PCR with
specific primers. Sample preparation is performed following the
protocol described in Table 1.
Cell Adhesion Assay for Leptospiral Proteins
175
43. Coomassie Brilliant Blue: 2.5 mg/mL solution. Weigh out
0.25 g Coomassie Brilliant Blue, dissolve in 10 mL of glacial
acetic acid, and add 1/1 (vol/vol) methanol and water to a
final volume of 100 mL. Stir the solution for 3–4 h and then
filter through filter paper. Store at room temperature.
44. Destaining solution: 10% acetic acid, 45% methanol. Mix
10 mL of glacial acetic acid and 45 mL of methanol and add
water to a final volume of 100 mL.
45. Dialysis membrane with a cutoff of 10 kDa.
2.3 Interaction Assay
1. ELISA microplates (96-well) for high binding.
2. Absorbance microplate reader.
3. 37
C incubator.
4. Microplate washer.
5. 50 mL tubes.
6. 1.5 mL vial.
7. Sealing tape for 96-well plates.
8. Wash buffer: 1Â PBS, 0.05% Tween 20 (PBS-T).
9. Blocking buffer: PBS-T, 10% dry milk or 1% BSA. Weigh out
10 g skimmed dry milk, and dilute the powder in 100 mL
PBS-T. For preparing BSA, weigh out 1 g BSA, and dissolve
in 100 mL of PBS-T.
10. Mouse antiserum against protein of interest.
11. HRP-conjugated anti-His monoclonal antibodies.
12. HRP-conjugated anti-mouse IgG.
13. Bovine serum albumin: 1 mg/mL in water. Aliquot and store
at À20
C.
14. Fetuin from fetal bovine serum: 1 mg/mL in water. Aliquot
and store at À20
C.
15. o-Phenylenediamine (OPD).
16. Hydrogen peroxide (H 2 O 2 ).
17. Citrate-phosphate buffer: 0.1 M citrate, 0.2 M phosphate,
pH 5.0.
18. Sulfuric acid: 2 M solution in water.
3 Methods
3.1 Cloning
and Expression
of Recombinant
Protein in E. coli
1. Genes of interest are amplified without the signal peptide (see
Note 7) from the genomic DNA of L. interrogans by PCR with
specific primers. Sample preparation is performed following the
protocol described in Table 1.
Cell Adhesion Assay for Leptospiral Proteins
175