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Biotinylation
NHS-PEO4-biotin (Pierce; MW 588.67), a typical amine-reactive N-hydroxysuccinimide (NHS)
ester, is shown below. The hydrophilic four-unit polyethylene oxide (PEO, also known as
polyethylene glycol or PEG) linker makes the compound water soluble and increases the
hydrophilicity of the adduct. This is the biotinylating reagent now in routine use in our lab.
O
H
N
N
H
PEO (PEG) linker
NHS
H
N
S
Biotin
O
O
O
O
O
O
O
N
O
O
Kinetic parameters are available for biotinylation of IgG with this reagent under a particular set
of reaction conditions—overnight in PBS pH 7.2 (0.15 M NaCl, 0.1 M NaH2PO4, pH adjusted to
7.2 with NaOH) at room temperature (Smith, G.P. Kinetics of amine modification of proteins.
Bioconjugate Chemistry, in press 2006). In these circumstances, the concentration of reagent
required to achieve a biotinylation level of m coupled biotins per molecule is given by the
formula

m 
m 


R  m  25.6  ln 1 
  P  2274  ln 1 

 128.9 
 128.9 

where R is the micromolar concentration of reagent required, P is the micromolar concentration
of IgG, and the desired modification level m is modest (less than ~10 coupled biotins per
molecule). The article cited above gives guidelines for extending the kinetics to other proteins.
Unfortunately, accurate empirical measurement of biotinylation level is difficult.
Standard biotin assays, which rely on competition between the analyte (the biotin to be
quantified) and another ligand for binding to avidin or streptavidin, give invalid results when the
biotin is coupled to a protein. A suitable protocol is described in the article cited above. For a
well-characterized biotinylating agent like NHS-PEO4-biotin, however, such quantitation should
be unnecessary: the biotinylation level actually achieved should not be far off what is predicted
in the equation above.
LARGE-SCALE BIOTINYLATION
In this exemplar protocol, 1 mg (6.7 nmol) of IgG in 90 µl PBS pH 7.2 is biotinylated to a level
of 3 biotins per molecule in a total reaction volume of 100 µl. If the protein were in some other
buffer, it would have to be equilibrated with PBS pH 7.2 by dialysis or some other method.
Classification of 1 mg as a “large amount” is somewhat arbitrary, but in this context three key
considerations are that this amount is enough to be measured by standard physico-chemical
methods such as scanning spectrophotometry, to manipulate freely without carrier protein to
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prevent loss by adsorption to vessel surfaces, and to carry out many rounds of affinity selection
(AffinitySelection.doc) and extensive binding assays (ELISA.doc and micropan.doc). Since the
final IgG concentration P will be 10 mg/ml = 67 µM during the biotinylation reaction, we
calculate from the formula above that the concentration of reagent R required to achieve a
modification level of m = 3 is 295 µM—a 4.4-fold molar excess over the IgG molecules.
Accordingly, a 2.95-mM dilution of the reagent—i.e., ten times the final concentration—will be
prepared and 10 µl will be added to the 90-µl protein solution.
1. Into a 1.5-ml microtube pipette 90 µl of IgG at a concentration of 11.3 mg/ml in PBS pH 7.2.
2. Pipette 100 µl dimethylsulfoxide (DMSO) into a Pierce No-Weigh tube containing 2 mg (3.4
µmol) NHS-PEO4-biotin, using the pipette tip to break through the foil seal; dissolve the oily
solid completely by pipetting up and down and scraping with the tip. (Although the reagent is
soluble in water, it is difficult to dissolve the oily solid in water directly, and significant
hydrolysis of the reagent can occur in the process; in contrast, the reagent is readily dissolved
and stable in dry DMSO, and the tiny concentration of the solvent in the final reaction mixture is
completely harmless to IgG or other proteins.)
3. Pipette the entire 100-µl DMSO solution into 1.05 ml PBS pH 7.2 in a 1.5-ml microtube, thus
making a 2.95-mM dilution; immediately vortex, pipette 10 µl into the microtube with the IgG,
vortex that microtube, microfuge it briefly to drive the solution to the bottom, and allow it to
react at least 4 h away from light. The molar ratio of reagent to IgG is 4.4. (The kinetic
parameters used to calculate the reagent concentration refer to room temperature incubations; the
parameters for incubation at 4ºC, say, are unknown, but in general might be somewhat different.
A 4-hr incubation is undoubtedly more than sufficient for the reaction to reach completion—i.e.,
for hydrolysis and modification together to have consumed all starting reagent.)
4. Into the reaction microtube pipette 1 ml TBS; vortex; transfer to a Centricon centrifugal
concentration device (Millipore) with a 30-kDa molecular weight cutoff; add an additional 700
µl TBS; remove uncoupled biotin by four cycles of concentration to ~50 µl according to the
supplier’s instructions and re-dilution to 1.8 ml; concentrate to ~50 µl one more time; add 400 µl
TBS to the final retentate, vortex the Centricon device to ensure all the protein is swept off the
membrane, and collect the diluted retentate by back-centrifugation according to the supplier’s
instructions; transfer the retentate into an appropriate vessel. [The Centricon device is suitable
for up to ~1 mg IgG, corresponding to a maximum concentration of ~20 mg/ml in the ~50-µl
deadstop (fully concentrated retentate). For larger amounts of protein, uncoupled biotin can be
removed by dialysis.]
5. The theoretical IgG concentration in the final ~450-µl solution from the above protocol would
be ~2.2 mg/ml assuming no losses. The concentration can be measured empirically
spectrophotometrically or by the Bradford reaction, but not by the BCA assay because of the
high concentration of Tris. A typical yield is ~80% of the starting IgG at the 1 mg scale, but can
approach 100% at larger scales.
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6. The final Bio-IgG preparation can be stored in the refrigerator for months; for long-term
storage, however, it is advisable to mix it with an equal volume of ultrapure glycerol and store it
at –20ºC, at which temperature the solution doesn’t freeze.
NOTE: The foregoing protocol can be scaled down at least to 500 µg and scaled up indefinitely,
using suitable dialysis and/or concentration devices to remove uncoupled biotin and exchange
buffer as needed. IgG concentrations from about 150 µg/ml (roughly the upper limit for the
small-scale protocol below) all the way to the solubility limit can be accommodated by adjusting
the reagent concentration according to the equation.
SMALL-SCALE BIOTINYLATION
In this protocol it is assumed that the concentration of IgG is no higher than ~1 µM (150 µg/ml)
in the final reaction mixture. At such low IgG concentrations, the first term on the right-hand
side of the formula above is negligible, and the concentration of reagent required to achieve a
biotinylation level of m = 3 is ~54 µM regardless of the IgG concentration. There is no sharply
demarcated lower limit on the IgG concentration, but at exceedingly low concentrations (less
than ~100 ng/ml) loss of IgG from adsorption to vessel walls might be substantial. Once
biotinylation is complete, however, a large amount of BSA carrier protein will be added to the
Bio-IgG, thus protecting it from any further losses due to adsorption. The final reaction volume
in the exemplar protocol below is 10 µl, consisting of 5 µl IgG and 5 µl 108-µM reagent (giving
the desired final reagent concentration of 54 µM). In those circumstances the upper limit on the
amount of IgG would be ~1.5 µg in 5 µl PBS pH 7.2, yielding ~1 µg of final Bio-IgG product—
enough for 3–4 rounds of affinity selection (AffinitySelection.doc), especially if selection is
carried out in ELISA wells rather than 35-mm Petri dishes.
7. Into a 500-µl microtube pipette 5 µl of IgG in PBS pH 7.2. The IgG concentration should be
no higher than ~300 µg/ml.
8. Pipette 100 µl DMSO into a Pierce No-Weigh tube containing 2 mg (3.4 µmol) NHS-PEO4biotin, using the pipette tip to break through the foil seal; dissolve the oily solid completely by
pipetting up and down and scraping with the tip (see comment at step 2 above).
9. Pipette 10 µl of the DMSO solution into 3.14 ml PBS pH 7.2 in a 4-ml test tube, thus making
a 108-µM dilution; immediately vortex, pipette 5 µl into the microtube with the IgG, vortex that
microtube, microfuge it briefly to drive the solution to the bottom, and allow it to react at least 4
hr at room temperature away from light.
10. Into the reaction microtube pipette 100 µl 1 M ethanolamine, pH adjusted to 9.0 with HCl;
vortex, microfuge briefly to drive solution to the bottom, and incubate in the cold for at least 1 h
away from light. The high concentration of primary amine quenches (reacts rapidly with) any
residual unhydrolyzed reagent, thus ensuring that the BSA to be added at the next step will not
be biotinylated.
11. Add 400 µl 2.5-mg/ml BSA in TBS, vortex, and transfer to a Centricon centrifugal
concentration device with a 30-kDa molecular weight cutoff ; add an additional 1.3 ml TBS;
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remove uncoupled biotin by four cycles of concentration to ~50 µl according to the supplier’s
instructions and re-dilution to 1.8 ml; concentrate to ~50 µl one more time; add 100 µl TBS to the
final retentate, vortex the Centricon device to ensure all the protein is swept off the membrane,
and collect the diluted retentate by back-centrifugation according to the supplier’s instructions;
transfer the retentate into a 500-µl microtube.
NOTE: The BSA acts as a carrier protein, blocking any further loss of Bio-IgG by adsorption to
vessel walls; it is added only after all residual unconsumed reagent is quenched in the previous
step. The total amount of BSA added, 1 mg, is fully soluble in 50 µl—the approximate minimal
volume achieved in each cycle of concentration on the Centricon device.
12. The final Bio-IgG preparation can be stored in the refrigerator for months; for long-term
storage, however, it is advisable to mix it with an equal volume of ultrapure glycerol and store it
at –20ºC, at which temperature the solution doesn’t freeze.
VARIATIONS
A. Just about any water-soluble protein can be substituted for IgG; the article cited at the
beginning of this document gives guidelines for extending the procedure to proteins other than
IgG.
B. There is no apparent upper limit to the amount of protein that can be subjected to the largescale procedure. The Centricon ultrafilters have a capacity of the order of 1 mg; if larger
amounts of protein are biotinylated, therefore use dialysis or some larger ultrafilter (for example,
Ultrafree 15 from Millipore) to dialyze away unreacted biotin and concentrate the conjugate.
C. Some selector proteins other than antibodies—particularly low-molecular-weight proteins—
might be significantly inactivated by modification to a level of 3 biotins per molecule. If you are
uncertain, it is advisable to under-biotinylate by reducing the target modification level well
below 3. In those circumstances, a substantial fraction of the selector molecules would be
expected to be conjugated to a single biotin—the mildest possible modification level. At the
same time, another substantial fraction will remain unconjugated. In “one-step” affinity
selection (AffinitySelection.doc), non-biotinylated selector should not interfere as long as the
streptavidin-coated dish or well is charged with an excess of biotinylated selector. In “two-step”
affinity selection, reaction of target phage with unconjugated selector (making a complex that
cannot be subsequently “captured” on a streptavidin-coated dish or well) competes against
reaction with conjugated selector (making a complex that can be captured); if target phage are
limiting, this side-reaction can reduce yield. In most cases, however, this reduction in yield is
not severe enough to compromise affinity selection significantly.