胶乳包被程序

1. Calculate the amount of each coparticleonent needed.Note: The Coupling Procedure MS

Excel Calculation Sheet may be utilized by placing “0” in the fields for EDAC:COOH.

2. Prepare and check all stock coparticleonents required.

3. Once the amount of each coparticleonentis prepared, set up the binding reaction by pipetting the followinginto microcentrifuge tubes in the order given:

a. 50 μL 500 mM stock MES buffer: 25 mM final

b. DI Water to make 1.0 mL final volume

c. 100 μL of 10.0% solids stock particles: 1.0% solids final

d. Protein stock solution: (the protein should be added last and mixed very rapidly into the reaction mixture by syringing repeatedly with the pipettor)

Note: Improper mixing can yield unevenlycoated particles.

4. Mix tubes at room teparticleerature on a mixing wheel or other device for

one hour.

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

5. Remove unbound protein: pellet particles by centrifugation and decant the supernatant.

6. Perform two washes with your buffer(this may be the MES buffer). Pellet particles by centrifugation and decant the supernatant. Resuspend pellets between washes using ultrasonication.

7. Resuspend final pellet to desired % solids with the same buffer. For exaparticlele: 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.

8. Perform the BCA Assay for particles as an analytical tool to assess the amount of protein bound on the particles.