TITLE: Covalent Coupling Protein to Carboxylated Microparticles via EDAC 
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details how to covalently couple proteins to carboxylated polystyrene particles (microspheres) via EDAC. In this reaction, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) serves as an agent for covalently coupling protein (e.g. peptides, IgG, etc.) to carboxyl-modified particles by forming an amide bond. Various 2-(</span><span style = "font-style:italic;">N</span><span>-morpholino)ethanesulfonic (MES) acid buffers are used to maintain the appropriate pH ranges. Glycine is used as a quencher to cease the reaction. Bovine serum albumin (BSA) is used as a blocking agent, to prevent non-specific interactions with and between particles (e.g. hydrophobic interactions).</span></div><div class = "text-block">This protocol's development was guided by the information contained in Bangs Laboraties Inc. Tech Note 205, Covalent Coupling. This file is attached as a PDF.</div></div>

[STEPS]
?. [Wash Particles]
Vortex particle mixture.Centrifuge at Remove supernatant.Resuspend in  Activation buffer.Repeat for a total of two washes; Do not resuspend in Activation Buffer the second time.
Centrifuge: 9900 34, 10 min
500 µl
All centrifugation times and speeds will depend on particle size. They will need to be adjusted accordingly.
?. [Wash Particles]
Add  particles to 2 mL centrifuge tube.Add  Activation buffer.
10 µl
500 µl
?. [Wash Particles]
Sonicate carboxylated polystyrene particle stock in sonicating bath for  .Vortex mix particle stock for .
?. [Bind EDAC to Particles]
During the centrifugation times in Step 3, make EDAC solution:Add  Activation buffer to  EDAC aliquot.
5 mL
5 mg
This amount of EDAC is in excess. Tech Note 205 uses solution with100 mg EDAC for 100 mg particles. The excess EDAC is mostly removed by centrifugation.
?. [Bind EDAC to Particles]
Resuspend particle pellet from Step 3 with EDAC/Activation buffer, ensuring that particles are well suspended.Agitate on orbital mixer,  .
?. [Bind EDAC to Particles]
Vortex particle mixture.Centrifuge at  .Remove supernatant.Resuspend in Coupling buffer.Repeat for a total of two washes.
Centrifuge: 9900 34, 10 min
?. [Couple Protein to Particle]
During the centrifugation times in Step 6, make Protein solution:Add protein for 1-10X coverage (as calculated below) to  Coupling buffer.
500 µl
The amount of protein (e.g. antibody, peptide) will depend on particle concentration, size, etc. The following equation is used to calculate the necessary amount (adapted from TechNote 205):S = \frac{6C}{ \rho d}S (mg protein / g particles): amount of protein for surface saturationC (mg protein / m2 particle surface): capacity of particle surface for a given proteinρ (g / cm3): density of particled (μm): mean particle diameter.
?. [Couple Protein to Particle]
Combine pellet suspension and Protein solution. Pipet mix.Agitate on orbital mixer, to    at room temperature.
?. [Couple Protein to Particle]
Centrifuge at  .Remove supernatant.Resuspend particle pellet with  Coupling bufferVortex mix.Centrifuge at  .
Centrifuge: 9900 34, 10 min
500 µl
Centrifuge: 9900 34, 10 min
?. [Quench Coupling Reaction]
Resuspend particle pellet from Step 9 with  Quenching solution.Agitate on orbital mixer,   at room temperature.
1 mL
?. [Incubate with Blocking Buffer (optional)]
Centrifuge at  .Remove supernatant.Resuspend particle pellet with  Blocking solution Agitate on orbital mixer,   at room temperature.
Centrifuge: 9900 34, 10 min
500 µl
?. [Wash and Resuspend]
Centrifuge at  .Remove supernatant.Resuspend particle pellet with  Storage buffer.Vortex mix.
Centrifuge: 9900 34, 10 min
500 µl
?. [Wash and Resuspend]
Centrifuge at  .Remove supernatant.Resuspend particle pellet with Storage buffer to desired storage concentration.Store at  .
Centrifuge: 9900 34, 20 min
4 °C