How to Biotinylate with Reprodicible Results
Introduction
The Biotin-Streptavidin system continues to be
used in many protein-based biological research
applications including; ELISAs, immunoprecipitation, Westen blotting, general immobilization and
detection, and many other biological procedures.
Although pre-biotinylated proteins are often available from commercial sources, there are many instances when specialized proteins are not available
in this form; thereby requiring the researcher to
biotinylate their own protein.
However, there are many common problems and
other pitfalls associated with standard biotinylation procedures, for example;
- A researcher is often uncertain a
reaction worked properly or even to
what degree
- Over-biotinylation often causes precipitation and loss of protein
- Over-biotinylation often reduces protein activity and/or function
For this reason, streptavidin-binding assays were
developed and used to quantify the degree of
protein biotinylation. For example, two such assays include the HABA and a FluoroReporter®
streptavidin binding assay. However, streptavidin
binding assays suffer from numerous shortcomings:
- High cost (laborious and timeconsuming)
- Require expensive, often unavailable
equipment (e.g. fluorimeter)
- Require external protein calibration
curve
- Binding assays are destructive in nature and consume significant quantities of often precious protein
- Binding assays almost always underrepresent the number of biotin molecules actually attached to the protein
How can these problems be solved?
- Control the amount of biotin on your
protein for assay optimization
In order to avoid over modification which
often causes precipitation and or greatly
reduces activity you should select a directly traceable biotinylation kit. Such a
kit would allow you to quickly determine
the amount of biotin incorporated into a
protein before every assay. Such traceable biotinylation would enable you to
quickly determine the number of biotin
molecules present on the protein and
thus allow a minimal amount of biotin
just enough to successfully complete an
assay without disrupting activity or function. A fast, reliable and easy to use method for determining the number of
biotins attached to a protein would eliminate the desire to move on blindly to the
next step of an often complicated down-stream assay.
- Biotin quantification without expensive
equipment and additional costly assays.
Many of the current methods for quantifying biotin on proteins require a secondary assay such as the HABA or the
FluorReporter® (Invitrogen) assay. The
former assay is a destructive assay that is
less sensitive and can consume up to 75
ug of the labeled protein in the assay. It
also requires an external streptavidin based calibration curve. The latter, FluoroReporter® assay, although more sensitive than the HABA assay, requires a
spectrofluorimeter or a fluorescent plate
reader. This assay also requires an external calibration curve. The destructive nature of the assays along with increased
labor cost and time diminish the general
implementation of these assays. Any
method for circumventing these limitations could be quite beneficial to any
small company or research lab that worries about the cost of such ancillary reagents and assays.
- Reproduce your results effectively.
Quick and accurate quantification of the
number of biotins incorporated in a protein will allow you to quantify your reactions each time you biotinylate. This
provides confidence in the quality of the
assay reagents being used. It also permits
quantitative comparison to previous biotinylation reactions. Quantifying the biotin MSR (biotin molar substitution ratio)
after labeling would allow you move on to
the next step of a process or assay with
much greater confidence.
- Choose a traceable biotin reagent with a
“built-in” signaling system.
When you choose the right biotin reagent, you will ensure tracking and identification of the entire labeling process,
always ‘dialing in’ and quantifying the
proper degree of biotin incorporation before every assay or process.
Current biotinylation products on the market that can help solve these problems:
Although there are several products on the market to address one or two of the solutions above,
SoluLink offers the only comprehensive solution
to all these problems in a single reagent. We also
provide easy to use automated calculators that
avoid any need to manually calculate how much
reagent to use or time consuming calculations.
SoluLinK also offers one-on-one technical support
for any biotinylation project and/or other conjugation support services. Solving all these problems with the use of a single reagent can help you
achieve your ultimate research goals while saving
you time, money, protein, and other valuable resources while providing valuable process information.
Control the amount of biotin on the protein
In order to address all of the common biotinylation problems, Solulink has developed ChromaLink Biotin (Figure 1). ChromaLink Biotin is a
water-soluble biotin labeling reagent with built-in
signal traceability that allows you to track and
rapidly calculate the exact number of biotins attached to a protein or antibody. The procedure
for labeling with ChromaLink Biotin is identical to
biotinylating with any other NHS-based biotinylation reagent. The key to solving the common
problems previously discussed revolve around the
unique, UV-traceable chromophore embedded within the linker itself. Following buffer exchange
of the labeling reaction, the biotinylated protein is
simply analyzed by measuring the A280 and A354
of the conjugate. Inserting the absorbance values
into a ChromaLink Biotin calculator (provided)
automatically calculates the final protein concentration and the number of biotins incorporated!
Biotin quantification using a simple spectrophotometer and no other costly reagents
Representative UV absorbance spectra of a biotinylated antibody using ChromaLink Biotin can be
used to illustrate how easy it is to quantify biotin
incorporation by a simple scan of the biotinylated
sample (Figure 2). Data can be acquired on any
conventional or NanoDrop® spectrophotometer
and the sample recovered after analysis (non-destructive).
Avoid Additional Costly Assays
In order to illustrate how the HABA assay (Pierce
Chemical Co., Rockford, IL) often under reports the
number of incorporated biotins versus the ChromaLink Biotin method, both assays for biotin incorporation were compared and results summarized in
Table 1. Data in the table was generated by biotinylating (500 ul @ 5 mg/ml) of a bovine IgG sample
at 5, 10, and 15 mole equivalents using ChromaLink
Biotin.
As seen from the results, HABA measurements
yield lower estimates of biotin incorporation resulting in significant differences between the two assays. For example, the biotin molar substitution
ratio calculated using the HABA dye-binding assay
is generally 1/3 the value obtained with the ChromaLink method. The HABA dye-binding assay generally underestimates the true biotin molar
substitution ratio because it measures the number
of moles of biotin available for binding to streptavidin and not the absolute number of biotin molecules attached to the antibody surface. For example, two biotin molecules in close proximity to
each other are likely to bind to a single streptavidin
molecule.
Label Reproducibility
Triplicate biotinylation reactions were set-up using
bovine IgG @ 5 mg/ml and 6 equivalents of
ChromaLink Biotin reagent. After purification, each
sample was scanned using a NanoDrop® spectrophotometer (Figure 3), and the resultant spectra
overlaid. As clearly demonstrated, results are easy
to confirm and reproduce, time after time.
Make Assay Optimization Simple
Direct ELISA
A goat anti-bovine IgG antibody was biotinylated
using ChromaLink® Biotin to obtain a series of different molar substitution ratios. The biotinylated
antibodies were then used to detect immobilized
antigen (bovine IgG) in a standard ELISA procedure.
Purified bovine IgG was immobilized (2-fold dilution series) (0.5 - 5,000 ng/ml). After immobilization (4 hr @ RT), wells were blocked with 1%
casein/PBS and subsequently washed. The plate was then incubated with streptavidin-HRP @ 1 µg/ml for 60 minutes. After washes, TMB substrate 3,3’,5,5’- tetramethylbenzidine) was added for 20 minutes. Signals were measured on a conventional plate reader @ 650 nm. Direct ELISA dose response curves were plotted as illustrated below
Results:
Signal/noise increased approximately 2.9 fold (linear portion of the curve) as the biotin MSR
increased from 1.3 to 6.1 as illustrated in Figure 4.
Background controls were constant across the various MSRs (data not shown).
To further illustrate the relationship between signal/noise and MSR for this antibody/antigen pair,
plots were generated at a single fixed antigen concentration (e.g.2 ng/well) across a range of molar
substitution ratios (Figure 5).
Results:
Measured signal/noise increases almost
2.9-fold as the MSR goes from 1.3 to 6.1. Note the
slight reduction in signal as the MSR goes beyond
6.1 probably due to over-modification of the antibody.
Conclusion:
You have more important things to
do than worry about biotinylating a protein.
ChromaLink Biotin allows you to biotinylate proteins quickly and easily and then confirm the
number of biotins incorporated so you can proceed with confidence!!
Recommended Products:
[B-9007-105K] ChromaLink® Biotin Labeling Kit
[B-9007-105K] ChromaLink® One-Shot Kit
[B-1007-110] ChromaLink® Labeling Reagent