胶乳包被一步法操作流程

One Step Coupling Procedure:

1. Calculate the amount of EDAC

required.Note: The Coupling Procedure MS Excel Calculation Sheet may be utilizedto perform the calculations.

Given Equations:

(Particle acid content) meq/g is equivalent to μmole/mg Note: 1 mL of 1% Particle contains 10 mg Particle

(Acid content, μmol/mg) (10 mg particles) (desired ratio) = μmol EDACrequired (μmol EDAC required)/(52μmol/mL) = mL EDAC stock per mL of reaction

2. Set up binding reaction by pipetting into microcentrifuge tubes in the order

given:

a. 500 mM stock MES buffer: 25 mM final

b. Water to make 1.0 mL fi nal volume

c. 10.0% solids stock particles: 1.0%solids fi nal

d. Protein stock solution (add last)

3. Mix the tubes for approximately 15 minutes on a mixing wheel at room

teparticleerature.

Note: Gentle, constant mixing is iparticleortant for microparticle reactions.

4. Prepare the EDAC solution immediately before use and mix the calculated

volume rapidly into the reaction by syringing repeatedly with the pipettor.

5. Mix tubes at room teparticleerature on a mixing wheel or other device for one hour. particles may cluparticle during this time, but this is not unusual or harmful.

6. Remove unbound protein: pellet particles by centrifugation for Carboxylate-

modifi ed Particle, or magnet for Sera-Mag Particle, and decant the supernatant.

7. Perform two washes with your buffer.(This may be the MES buffer or a higher pH buffer of your choice.) Pelletparticles by use of a centrifuge for Carboxylate-modifi ed Particle, or magnet for Sera-Mag Particle, and decant the supernatant. Resuspend pellets

between washes by ultrasonication.

8. Resuspend fi nal pellet to desired % solids with buffer that does not contain

blocking proteins. (This may be the MES buffer or a higher pH buffer of your choice. For example: If the target % solids is 1.0%, then one would add 0.97 mL of the same buffer, given that some liquid remains after pellet formation.