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HRP Chemiluminescence ELISA-P

A. Solutions and Reagents

  1. Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/l NaN3 (pH 9.6). Use 1 µM synthetic peptide in carbonate buffer.
  2. 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.
  3. Wash Buffer: 1X PBS containing 0.05% Tween® 20 (PBST)
  4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
  5. Primary Antibody Dilution Buffer: 1 mg/ml BSA in PBST
  6. Secondary Antibody Dilution Buffer: 3% BSA in PBST
  7. 96-Well Plate: Solid white or opaque plates are recommended for chemiluminescent detection.

B. Binding Peptides to 96-Well Plate

  1. 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.
  2. Wash plate three times 200 µl/well with wash buffer.
  3. 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

  1. 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.
  2. Wash five times with wash buffer.
  3. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (# 7003) and Stable Peroxide Buffer.
  4. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1–10 minutes following addition of the substrate.
  5. Optimal signal intensity is achieved when read within 10 minutes.

D. Protocol for HRP-Conjugated Secondary Antibody

  1. 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.
  2. Wash three times with wash buffer.
  3. 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.
  4. Wash five times with wash buffer.
  5. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution (# 7003) and Stable Peroxide Buffer.
  6. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425nM within 1–10 minutes following addition of the substrate.
  7. Optimal signal intensity is achieved when read within 10 minutes.

*Recommended HRP-linked Secondary Antibodies:

posted October 2010
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