Polymer Laboratories Particle Technologies Technical Information

Clinical Diagnostic Particles TECHNICAL BULLETIN

L002: COVALENT COUPLING OF A PROTEIN TO POLYSTYRENE LATEX

This technical bulletin is written solely as a guide for the covalent coupling of a protein to polystyrene latex. Variations may be required for particular systems and optimization is recommended prior to use on a large scale.

PREPARATION

CARBONATE BUFFER	0.1M Na₂CO₃ added to 0.1M NaHCO₃ until pH=9.6
PHOSPHATE BUFFER	0.02M Na₂HPO₄ added to 0.02M NaH₂PO₄ until pH=4.5
CARBODIIMIDE	carbodiimide in phosphate buffer at 2%
BORATE BUFFER	0.1M boric acid adjusted to pH 8.5 with 1M NaOH
ETHANOLAMINE	ethanolamine in borate buffer at 15µl/ml
BSA BUFFER	BSA dissolved in borate buffer at 10mg/ml
STORAGE BUFFER	PBS containing 10mg/ml BSAm, 0.1% NaCI and 5% glycerol

PROCEDURE

1	Dilute 0.5ml PL-Latex SuperCarboxyl (10% solids) with ca. 5 ml carbonate buffer
2	Centrifuge to a pellet and discard the supernatant
3	Resuspend in carbonate buffer
4	Centrifuge to a pellet, discard the supernatant and resuspend in phosphate buffer
5	Repeat step 4 twice
6	Centrifuge to a pellet, discard the supernatant and resuspend in 2.5ml phosphate buffer
7	Add 2.5 ml carbodiimide and mix gently for 3.4 hours
8	Centrifuge to a pellet, discard the supernatant and resuspend in borate buffer
9	Repeat Step 8
10	Add 1-2 mg of protein and mix gently overnight at ambient temperature
11	Add 0.2ml ethanolamine, centrifuge to a pellet and discard the supernatant
12	Resuspend in BSA buffer, vortex and mix gently for ca. 30 minutes
13	Centrifuge to a pellet, discard the supernatant and resuspend in BSA buffer
14	Centrifuge to a pellet, discard the supernatant and resuspend in storage buffer

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