Tetramer Preparation: Preparation and Storage of Biotinylated Monomer Stocks

Reagents

PBS Buffer

PBS Buffer exchange:

  1. Collect sample fractions and centrifuge.

    From the MonoQ figure, collect the correct fractions and pour them into an ultrafree-4 centrifuge filtering device (MWCO: 10k).  Centrifuge at 3080g at 4(C until the entire collected sample volume is below 1ml. 

  2. Add PBS Buffer and centrifuge.

    Add the PBS Buffer to the ultra-free centrifuge filtering device and mix by inversion.  Centrifuge until the sample volume is below 500 µl.

  3. Repeat the afore-described buffer exchange once.
  4. Transfer the sample to an eppendorf tube and store on ice or at 4(C.
  5. Take UV scan of the sample.

    Dilute a sample of the monomer by 1:50 or 1:100 (final volume: 100 µl) and take a UV scan from 240nm to 320nm.

  6. Calculate the concentration of monomers.

    This can be done by entering the values from the UV scan for 260, 280 and 320 AU into the appropriate sections of the Altman tetramer database.  The database will calculate the amount of PBS buffer to be added to the monomer sample to achieve a concentration of 2mg/ml.

  7. Dilute the monomer sample to 2 mg/ml with PBS buffer.
  8. Aliquot the monomers to 100 µl fractions.
  9. Quick freeze the monomers and store at -80 °C.

    Use liquid nitrogen to quick freeze the monomers.  Enter the location of the monomers into the "Tetramer Stocks" database.