Purifying antibodies using random peptides on TentaGel Beads in Column Protocol

2015-01-13

azim58 - Purifying antibodies using random peptides on TentaGel Beads in Column Protocol

Purifying antibodies using random peptides on TentaGel Beads in Column
Protocol
(Protocol adapted from Current Protocols in Immunology 9.3 Basic Protocol 3)

Materials
TentaGel beads with synthesized peptide

* Note that an aliquot can be taken to Life Sciences for N Terminal
  sequencing to verify the identity of the peptide if desired. John Lopez
  runs the sequencer in the Protein core he will need very little
  material 2-3 beads. It will cost $5 per residue.
* Zhao mentinoed that it may be better to use Sephadex beads rather
  than Tentagel beads for antibody purification
Column

* disposable 5 mL polypropylene columns (prod # 29922 Thermo)
* 5 mL plastic columns at cabinet at left side of scanner room door
Equilibration buffer

* current protocols suggests: 50 mM potassium phosphate (pH 7.5)/0.4 M
  NaCl with and without azide
* Bart and Kaitlyn have used TBST. Bart mentioned that I'll have higher
  background without TBST.
* Shen uses 21001 Protein A IgG Binding Buffer
Antiserum known to contain anti-peptide antibodies
Neutralization buffer

* current protocols suggests: 1 M Tris⋅Cl, pH 7.5
* Bart and Kaitlyn have used 1.5 M Tris 150 mM NaCl pH8 before
  o Tris MW 121.14 g/mol
  o Tris: 1.5 mol/L*121.14g/mol*1L = 181.71 g Tris
  o NaCl: 150 mmol/L*1*mol/(1000*mmol)*58.5*g/(1*mol)*1*L= 8.78 g NaCl
  o about 50 mL of 6 N HCl was needed to reach pH 8

Elution buffer (choose one of the following):

* Acidic elution buffer: 0.1 M glycine (pH 2.8)/0.15 M NaCl, 0.1 M
  acetic acid, or 0.1 M sodium citrate, pH 3.0 (see recipes)
  o Bart has used 0.1 M citric acid pH 2.3
  o glycine: 0.1*mol/L*75.07*g/mol*1*L = 7.507 g glycine
  o 150 mM NaCl -> 8.78 g NaCl
  o about 5 mL 6 N HCl required to reach pH 2.8

* Basic elution buffer: 0.1 M diethanolamine, pH 9.8 (see recipe)
* Chaotropic elution buffer: 4 M sodium isothiocyanate, pH 7.5, 6 M
  urea, pH 7.5,
* 5 M guanidine⋅HCl, pH 7.5 (see recipes)
* Shen uses 21004 IgG Elution Buffer
PBST
0.45-μm syringe filter with 3- or 5-ml syringe with Luer lock (needed if
you plan to "clarify", but this may not be necessary)
Slide-A-Lyzer for dialysis
100, 70, 50, and 30% DMF

* Don't use polystyrene or polycarbonate with DMF. You can use glass
  and polypropylene with DMF. Most pipette tips are polypropylene.
  Generally plastic that is completely clear cannot be used with DMF, but
  if it is slightly opaque then it is often okay.
Protein-lo bind tubes (cat no 022431081 from Eppendorf (can be ordered
through VWR or many others))

Procedure

* If the peptide has just been synthesized onto the beads, gradually
  equilibrate the beads from an organic solution to an aqueous solution
  o May want to make a 20% bead slurry in DMF.
  o May want to use 100 uL of beads
  o Prepare 100, 70, 50, 30 % DMF (10 mL each) if these solutions are
    not prepared already
    - DMF last seen in chemical hood (B225 A)
    - 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
    - I think polycarbonate is okay
    - Any waste DMF should go in organic waste

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

- Wash beads with 5 mL PBS

* Inspect the affinity column. If it shows signs of microbial growth,
  discard it. If it has a few air bubbles, unpack and repack it.
* Pre-equilibrate the column by washing with equilibration buffer (such
  as TBST) until the monitored absorbance (i.e., fractions or online UV
  monitor) reaches a baseline. Alternatively, it is probably fine to flow
  a set volume through such as 5 mL. Note that if the column was just
  washed with DMF and then PBS this step is probably not necessary.
  o The column may be run either under gravity or with a pump. For
    best results with a
    1-cm-diameter column, keep the flow rate for all steps below 1
    ml/min. Approximately 1
    drop per second is good.
    Antibodies absorb well in the near UV region, so monitoring anywhere
    in the range of 254
    to 280 nm is acceptable. The baseline should level off at <0.1 AU.

* Prepare the sample by adding 1 to 2 ml antiserum known to contain
  anti-peptide antibodies to an equal volume equilibration buffer (such
  as TBST). Another dilution might be reasonable as well. Bart recommends
  a 1 to 10 or 1 to 100 dilution (1 to 100 dilution may be too small for
  many applications).
  o While dilution is not absolutely necessary for effective use of
    this protocol, it decreases viscosity and improves flow.

* Clarify diluted serum sample
  o For clarification using a syringe filter: Pass the diluted serum
    sample through a
    0.45-μm syringe filter attached to a 3- or 5-ml syringe with Leur
    lock.
    - Removing particulate material from the sample improves flow
      and prolongs column life.

o For clarification by centrifugation: Centrifuge 10 minutes at
    1000 × g, room temperature.

* Apply the sample to the column. Collect the antiserum that flows
  through unbound. Note that it may be a good idea to incubate the sample
  with the column on the rotisserie for 1 to 2 hr with the column capped
  and with parafilm on the bottom. Alternatively an incubation on the
  rotissery at 4 C overnight may result in even more cognate specific
  binding. Later on remove the top cap, and then the bottom parafilm
  o An alternative is to load the sample onto the pre-equilibrated
    affinity resin in batch, instead
    of in a column. In this case, combine the sample with 3 to 4 ml resin
    in a tube and gently
    tumble overnight at 4°C. Pour the resin into the column and continue
    with step 6. This
    alternative allows unattended extended sample loading for convenience.
    Typically, the antiserum contains a high concentration of
    anti-peptide antibodies; thus,
    antiserum that flows through the column still contains a significant
    amount of useful
    antibodies. For economy, this flow-through may be saved and
    rechromatographed.

* May want to collect unbound antibody for storage since there could be
  some valuable antibodies in this solution.
* Wash the column
  o current protocols suggestion: Wash the column with equilibration
    buffer (such as TBST), monitoring the eluent until UVabsorbance
    returns to baseline.
    - Use at least 20 ml potassium equilibration buffer (such as
      TBST) for this step.

* Prepare for elution collection
  o current protocols suggestion: Load ten test tubes (best to use
    protein-lo bind tubes to prevent the small amount of antibody present
    from sticking to the sides of the tube) with 0.2 ml neutralization
    buffer if acidic or basic elution is to be performed, or water if
    chaotropic elution is to be used.
    - See Troubleshooting, Problematic Affinity Chromatography for
      a discussion of elution
      method.

* Elute bound antibody
  o current protocols suggestion: For acidic elution: Elute the
    column with 5 ml acidic elution buffer. Collect 1-ml
    fractions directly into the tubes containing neutralization buffer.
    - Although not usually necessary, as soon as they are eluted,
      the antibodies may be swirled
      gently to mix them thoroughly with the dense neutralization buffer
      in the tube.

* Nanodrop the elution fractions to determine which ones contain
  antibody
* Pool fractions containing antibody
* May want to save some non-dialyzed sample
* Perform dialysis on collected antibody solution
  o current protocols suggestion: dialyze (APPENDIX 3H) against at
    least two
    changes of 100-fold excess volume at 4°C PBST. Aliquot as desired
    and store the
    antibody solution at −70°C.
    - Usually only three to five fractions will be pooled, since
      the flanking fractions contain
      minimal antibody.

o use Slide-A-Lyzer to perform dialysis

* Re-equilibrate the column
  o current protocols suggestion: by washing it with 20 ml
    equilibration buffer (such as TBST) with
    or without azide, depending on if the column is to be used again
    immediately or stored
    at 4°C.
    - To prolong column life, re-equilibrate it as soon as possible
      after elution. To prevent
      microbial contamination, store the column refrigerated in
      equilibration buffer with
      azide. Depending on the sample, and elution and storage conditions,
      expect 3 to 20 useful
      runs, perhaps more.

Questions about protocol
What type of column will I use?
plastic 5 mL column

How many Tentagel beads will I add to the column?
if you have hyperimmune sera, use all of them (~0.5 mL) if you do not
have hyperimmune sera or low titer sera use less maybe 100 ul but the
problem is smaller volumes mean higher flow rate through the column so
you would have to batch bind/batch elute.

What if I want to use less than 1 to 2 mL serum? Is this okay?
Bart:
You can use any volume you want. You would just use a smaller volume
during the loading step, still keep it at 1:10 to 1:100.

Should I use a pump to pass my solution through the column?
Maybe I'll just try gravity at first since it is simpler

Troubleshooting Protocol 062912
See Troubleshooting, Problematic Affinity Chromatography for a discussion
of elution
method.

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