Amine EviTag Conjugation to Primary Amine

Purpose

For conjugation of T2-MP EviTags Amine Functionalized quantum dots to peptides, proteins, and antibodies.


Materials Required

  1. 2nmol Amine Functionalized EviTags
  2. BS3, Bis(sulfosuccinimidyl) suberate (Pierce, part #21580)
  3. 10X PBS
  4. dH2O
  5. 2 - 100K Microspin Filters (Pall Corporation, part # OD100C33)
  6. 1 mg biomolecule of interest - customer supply
    • Note: biomolecule of interest must contain a terminal primary amine group and be free of preservatives, such as BSA and azide. Biomolecule solution must be amine free (1X PBS is recommended)
  7. 1 Disposable Plastic Column (Pierce part # 29922)
  8. 2 column frits (for top and bottom of column)
  9. 6ml Superdex200 Resin (GE Healthcare, part #17-1043-10 for large proteins and peptides) or Superdex30/75 Resin (GE Healthcare, part # 17-0905-10 or 17-1044-10 for small proteins and peptides)

Procedure

  1. EviTag Activation
    1. Activate 2.0 nmol of EviTag by adding 25µL 10mM BS3. and 25 uL 10X PBS. Bring final volume to 250 uL with dH2O.
    2. Incubate for 30 minutes.
    3. Desalt using a PD-10 column, eluting with 1X PBS. Collect colored portion.
  2. Biomolecule Coupling
    1. Add 1mg of biomolecule of interest.
    2. Incubate at room temperature for 2 hours under continuous, gentle shaking.
  3. Conjugate Purification
    1. Concentrate the conjugate to a total volume of ~200µl by centrifugation with 100K Microspin filter at 6,000 rpm for 5-10 minutes.
      Note: refer to manufacturer's directions for spin column use
    2. Wash the conjugate over the 100K MicroSpin filter 2 times with dH20.
    3. Purify the conjugate by HPLC* (recommended) or by size exclusion with Superdex** resin
      *For Superdex Column Purification:
      1. Pre-equilibrate the column with dH2O.
      2. Load the concentrated coupling mix onto the column and allow the material to enter the column bed.
      3. Elute under blacklight excitation with dH2O and collect the fluorescent fractions.
      4. Pool the fluorescent fractions and concentrate the conjugate to a total volume of ~100µl by centrifugation with 100K Microspin filter at 6,000 rpm for 5-10 minutes.
      5. Store conjugate at 4°C.

    *Recommended HPLC Column:
    TOSOH Bioscience
    TSK-Gel G4000SWxL HPLC Column
    #08542

    **Superdex200 Column Preparation:
    1. Pour ~5 ml of Superdex200 resin into an empty disposable plastic column with bottom frit fitted (avoid making bubbles).
    2. Allow resin to settle 10-20 minutes.
    3. Apply top frit.
    4. Proceed to step 3c above.
  4. Conjugate Characterization
    1. Record the optical density (OD) of the fractions at the EviTag exciton peak wavelength as well as at 260nm (for nucleic acid) or at 280nm (for protein).
    2. Record the fluorescence of the fractions at the EviTag emission peak wavelength (indicated on EviTag vial).
    3. Plot the OD readings and fluorescence values at each wavelength vs. the fraction number to generate elution profiles. The profiles can be used to identify which fractions contain EviTag conjugate and which contain free biomolecule. (Refer to Graph A)
      EviTag conjugate fractions will be represented by peaks with overlapping fluorescence and absorption spectra.
      Free biomolecule fractions will be represented by peaks with no fluorescence, but absorption at 260nm (for nucleic acid) or at 280nm (for protein).
      Graph A
    4. Pool the fractions containing free biomolecule and quantify amount of unreacted biomolecule (biomolecule out) using standard assay (e.g., Beer's Law; Lowry or Bradford assays for protein).
    5. Pool the fluorescent fractions for use in application.
    6. Determine the coupling efficiency using the following equation:
    7. 100 - [(mg biomolecule out) / (mg biomolecule in) x 100] = percent bound biomolecule

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