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Covalent Coupling of Proteins to IDC UltraClean Aliphatic Amine Latex(AML)

Coupling of the primary carboxyl groups of protein molecules to amino groups on a latex particle using a water soluble carbodiimide (WSC) is a common procedure. The reaction proceeds rapidly through an active ester intermediate.

The following procedure outlines both a simple one step method and a two step method that we have used with excellent results for coupling avidin, streptavidin, BSA, other proteins, and antibodies to aliphatic amine latex. The protocol is for a 1µm aliphatic amine latex at 1% solids. This reaction can easily be scaled up or down to fit individual needs. For example if a different particle size is used then the amounts of WSC and antibody (Ab) can be calculated from :-

For example we suggest for a 1µm particle you use 5mg of protein for 100mg of particles.

For 100mg of 0.3µm aliphatic amine particles we would need :-

And for 100mg of 4.3µm aliphatic amine particles we would need :-

Buffers & Reagents
MES Buffer 0.025 M pH 6
PBS Buffer 0.1 M pH 7.2
Storage Buffer 0.1 M PBS pH 7.2; 0.1% glycine; 0.1% NaN3
WSC EDAC (1 Ethyl 3-(3-Dimethyl Amino Propyl) Carbodiimide HCl) 50 mg/ml in MES
Antibody Ab of your choice at a concentration of 1.7mg/ml in MES
UltraClean Aliphatic Amine Latex As supplied at 4% solids.

Protocol

Preparation of the Latex
  1. Pipette 2.5 ml (40 mg/ml) aliphatic amine latex microspheres and dilute with 10 mL MES buffer.
  2. Centrifuge the mixture to sediment the particles; ~3,000G for ~20 min.
  3. Remove supernatant and re-disperse the pellet in 10 ml MES buffer.
  4. Centrifuge again and remove the supernatant from the particles.
  5. Re-suspend pellet in 5 ml MES buffer, being sure to completely suspend microsphere particles.
The latex suspension is now at approx. 2 % solids (i.e. ~20mg/ml).
  1. Pipette 2.5 ml (40 mg/ml) aliphatic amine latex microspheres and dilute with 10 mL MES buffer.
  2. Centrifuge the mixture to sediment the particles; ~3,000G for ~20 min.
  3. Remove supernatant and re-disperse the pellet in 10 ml MES buffer.
  4. Centrifuge again and remove the supernatant from the particles.
  5. Re-suspend pellet in 5 ml MES buffer, being sure to completely suspend microsphere particles.
The latex suspension is now at approx. 2 % solids (i.e. ~20mg/ml).

Coupling via a Two Step Procedure

  1. To 5ml of the protein solution in MES buffer, add 2ml of 50mg/ml EDAC in MES (freshly prepared)
  2. Incubate protein/EDAC mixture with gentle mixing at room temperature for 15 min.
  3. Take 5ml of the latex in MES buffer solution and add to the 5ml of protein solution.
  4. Incubate latex/protein mixture with gentle mixing at room temperature for 3 to 4 hours.
  5. Centrifuge to sediment the coated latex.
  6. Remove and retain supernatant for protein determination*.
  7. Re-suspend the pellet in 10 ml PBS.
  8. Centrifuge again to sediment the particles.
  9. Repeat steps 7-8 twice more for a total of 3 washes.
  10. Re-suspend the final latex in 10 ml Storage Buffer giving a final concentration of 1% solids. Store at 4°C until used. Do not freeze.

* At this step, the supernatant can be reserved and a protein determination made. Any residual protein in the supernatant can be subtracted from the original amount added. The remainder is coated on the particles. The only method for protein determination compatible with latex microspheres is the Micro BCA Protein Determination Kit from Pierce.

The NaN3 is present in the storage buffer as a biocide. If the latex is kept sterile, NaN3, which is not compatible with cell or tissue culture, can be omitted.

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