5. Mannan from Saccharomyces cerevisiae.
6. Bovine serum albumin (BSA).
7. Primary antibodies: anti-human C3 or anti-human C4produced in rabbit, goat, or sheep.
8. Secondary antibodies: HRP-conjugated antibodies with specificity for the antibody species and isotype of the primary antibody being used (e.g., anti-rabbit IgG-HRP, anti-goat
IgG-HRP, or anti-sheep IgG-HRP).
9. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes.
10. Bacterial counting chamber [cell depth: 0.02 mm
(1/50 mm)].
11. Water bath/dry block incubator.
12. 0.22 μm sterile syringe filters.
13. Spectrophotometer with a filter for 492 nm wavelength.
14. A kit to determine total protein concentration.
15. ELISA plates.
16. Citrate buffer: 0.1 M Na 3 C 6 H 5 O 7 , 0.2 M NaH 2 PO 4 , pH 5.0.
17. o-Phenylenediamine dihydrochloride (OPD): 0.05% solution
in citrate buffer.
18. H 2 O 2 : 0.015% solution in citrate buffer.
19. Wash buffer: PBS, 0.05% Tween (PBS-T).
20. Blocking buffer: PBS-T, 3% BSA.
21. Carbonate buffer: 100 mM NaHCO 3 , 100 mM Na 2 C0 3 ,
pH 9.6.
22. Alternative Pathway (AP) buffer: 0.96 mM sodium barbital,
144 mM NaCl, 2.48 mM barbituric acid, 1.4 mM MgCl 2 ,
10 mM EGTA, pH 7.3–7.4.
23. Gelatin Veronal Buffer (GVB
++ ): 0.96 mM sodium barbital,
144 mM NaCl, 2.48 mM barbituric acid, 0.83 mM MgCl 2 ,
0.25 mM CaCl 2 , pH 7.3–7.4.
2.5 SDS
Polyacrylamide Gel
1. Apparatus for SDS-PAGE electrophoresis (migration cell and
power supply).
2. Resolving gel buffer: Tris–HCl, pH 8.8. 1.5 M Tris Base
(187 g); add circa 900 mL ddH 2 O, stir the solution at least
10 m, adjust pH to 8.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
3. Stacking gel buffer: Tris–HCl, pH 6.8. 0.5 M Tris Base
(60.5 g); add circa 900 mL ddH 2 O, stir the solution at least
10 m, adjust pH to 6.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
Complement Resistance Assays
191
6. Bovine serum albumin (BSA).
7. Primary antibodies: anti-human C3 or anti-human C4produced in rabbit, goat, or sheep.
8. Secondary antibodies: HRP-conjugated antibodies with specificity for the antibody species and isotype of the primary antibody being used (e.g., anti-rabbit IgG-HRP, anti-goat
IgG-HRP, or anti-sheep IgG-HRP).
9. Refrigerated centrifuge with appropriate adaptors for 50 mL
tubes.
10. Bacterial counting chamber [cell depth: 0.02 mm
(1/50 mm)].
11. Water bath/dry block incubator.
12. 0.22 μm sterile syringe filters.
13. Spectrophotometer with a filter for 492 nm wavelength.
14. A kit to determine total protein concentration.
15. ELISA plates.
16. Citrate buffer: 0.1 M Na 3 C 6 H 5 O 7 , 0.2 M NaH 2 PO 4 , pH 5.0.
17. o-Phenylenediamine dihydrochloride (OPD): 0.05% solution
in citrate buffer.
18. H 2 O 2 : 0.015% solution in citrate buffer.
19. Wash buffer: PBS, 0.05% Tween (PBS-T).
20. Blocking buffer: PBS-T, 3% BSA.
21. Carbonate buffer: 100 mM NaHCO 3 , 100 mM Na 2 C0 3 ,
pH 9.6.
22. Alternative Pathway (AP) buffer: 0.96 mM sodium barbital,
144 mM NaCl, 2.48 mM barbituric acid, 1.4 mM MgCl 2 ,
10 mM EGTA, pH 7.3–7.4.
23. Gelatin Veronal Buffer (GVB
++ ): 0.96 mM sodium barbital,
144 mM NaCl, 2.48 mM barbituric acid, 0.83 mM MgCl 2 ,
0.25 mM CaCl 2 , pH 7.3–7.4.
2.5 SDS
Polyacrylamide Gel
1. Apparatus for SDS-PAGE electrophoresis (migration cell and
power supply).
2. Resolving gel buffer: Tris–HCl, pH 8.8. 1.5 M Tris Base
(187 g); add circa 900 mL ddH 2 O, stir the solution at least
10 m, adjust pH to 8.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
3. Stacking gel buffer: Tris–HCl, pH 6.8. 0.5 M Tris Base
(60.5 g); add circa 900 mL ddH 2 O, stir the solution at least
10 m, adjust pH to 6.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
Complement Resistance Assays
191