Purifying antibodies using random peptides on TentaGel Beads in Column Protocol
2015-01-13azim58 - 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
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
Column
- disposable 5 mL polypropylene columns (prod # 29922 Thermo)
- 5 mL plastic columns at cabinet at left side of scanner room door
- current protocols suggests: 50 mM potassium phosphate (pH 7.5)/0.4 M
- Bart and Kaitlyn have used TBST. Bart mentioned that I'll have higher
- Shen uses 21001 Protein A IgG Binding Buffer
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: 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
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
- 5 M guanidine⋅HCl, pH 7.5 (see recipes)
- Shen uses 21004 IgG Elution Buffer
- 45-μm syringe filter with 3- or 5-ml syringe with Luer lock (needed if
Slide-A-Lyzer for dialysis
100, 70, 50, and 30% DMF
- Don't use polystyrene or polycarbonate with DMF. You can use glass
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
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,
- Pre-equilibrate the column by washing with equilibration buffer (such
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
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
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
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
- Wash the column
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
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
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
- Pool fractions containing antibody
- May want to save some non-dialyzed sample
- Perform dialysis on collected antibody solution
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
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.