Example purification of antibodies with random peptides on tentagel beads 120611

2015-01-13

azim58 - Example purification of antibodies with random peptides on
tentagel beads 120611

* Prepared 100, 70, 50, 30 % DMF (10 mL each)
  o DMF last seen in chemical hood (B225 A)
  o DMF reacts with polystyrene transfer pipettes so use glass if
    possible. I used a glass aspirator pipette from box next to pH meter
    connected to a bulb
  o I think polycarbonate is okay
  o Any waste DMF should go in organic waste

* Dissolve beads with peptide in organic solvent such as DMF and
  gradually move them into an aqueous solution
  o Add 10 mL DMF to column supported by equipment to hold the tube
    up and let the liquid drain off into a waste container
  o Add 10 mL 70% DMF. Wait for it to drain. Then add 50% DMF. Wait.
    Then add 30% DMF. Wait
    - draining takes some time

o Wash beads with 5 mL PBS
  o Cap bottom. Add 3 mL PBS and 1 mL sera (1:4 dilution)
  o Cap column and rotate on rotissery for 2 hr at room temperature.
  o There was an inconsistent amount of reactivity with the beads.
    Perhaps the amount of beads pipetted was inconsistent since the beads
    settled so quickly

Reattempt at purifying antibody with peptide on 12-7-11

* Removed cap from bottom and allowed liquid to drain out
* resuspended in 5 mL PBS
* took aliquot to Life Sciences for N terminal sequencing (Bart did
  this)
* added 3 mL PBS and 1 mL sera (1:4 dilution)
* Capped column and rotated on rotissery 2 hr RT
* took column off rotissery and allowed beads to settle
* drained liquid off into container we saved
* added 5 mL PBS which drained into a new tube
* Added 95 mL PBS which drained into a new 50 mL conical (had to add
  the PBS in many 5 mL aliquots)
* Labeled 12 fraction 1.5 mL tubes
* Elution buffer: 0.1 M citric acid pH 2.3 filtered 4/18/11. This was
  from protein A protocol from purifying IgG
* Added 500 uL elution buffer per fraction
* washed column with 10 mL PBS
* Capped added 1 mL PBS
* Capped parafilmed. Stored everything at 4 C
* Nanodropped fractions. The 7th and 8th fractions appeared to have
  something in them maybe

azim58wiki: