A. Solutions and Reagents
- Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/l NaN3 (pH 9.6). Use 1 µM synthetic peptide in carbonate buffer.
- 10X Phosphate Buffered Saline (PBS): To prepare 1 l add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 l dH2O. Adjust pH to 7.4.
- Wash Buffer: 1X PBS containing 0.05% Tween 20 (PBST)
- Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
- Primary Antibody Dilution Buffer: 1 mg/ml BSA in PBST
- Secondary Antibody Dilution Buffer: 3% BSA in PBST
- 96-Well Plate: Solid white or opaque plates are recommended for chemiluminescent detection.
B. Binding Peptides to 96-Well Plate
- Coat the wells of a 96-well microtiter plate with 100 µl of 1 µM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hours at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
- Wash plate three times 200 µl/well with wash buffer.
- Block plate with 200 µl/well blocking buffer for 1 hour at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)
C. Protocol for HRP-Conjugated Primary Antibody
- Prepare recommended dilution of HRP-conjugated primary antibody with primary antibody dilution buffer. Add 100 µl to wells and incubate at 37°C for 1 hour.
- Wash five times with wash buffer.
- Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (# 7003) and Stable Peroxide Buffer.
- Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1–10 minutes following addition of the substrate.
- Optimal signal intensity is achieved when read within 10 minutes.
D. Protocol for HRP-Conjugated Secondary Antibody
- Prepare recommended dilution of primary antibody with primary antibody dilution buffer. Add 100 µl to wells and incubate overnight at 4°C or 2 to 6 hours at 37°C.
- Wash three times with wash buffer.
- Prepare recommended dilution of HRP-conjugated secondary antibody with secondary antibody dilution buffer. Add 100 µl to wells and incubate at 37°C for 1 hour.
- Wash five times with wash buffer.
- Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (# 7003) and Stable Peroxide Buffer.
- Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1–10 minutes following addition of the substrate.
- Optimal signal intensity is achieved when read within 10 minutes.
*Recommended HRP-linked Secondary Antibodies:
posted October 2010