How to Maximize the Capture of Biotinylated Targets with Streptavidin Magnetic Beads
Introduction
The use of immobilized streptavidin on
microwell plates and beads both non-magnetic
and magnetic is prolific in scientific assays. The
binding constant of biotin to streptavidin, >10-15
M-1, is near covalent and the kinetics of binding
is extremely fast making the biotin-streptavidin
pair ideal for bioassays on streptavidin
immobilized surfaces. It has been shown that
biotinylated biomolecules (i.e. antibodies,
peptides, and oligonucleotides) immobilized on
streptavidin surfaces retain their binding
affinity.
Streptavidin-coated magnetic beads, with their
fast magnetic response time, have particular
utility due to their ease of processing, leading to
their use in many high throughput and
multiplexed assays. Steptavidin-coated
magnetic beads have been incorporated in
automated platforms such as the KingFisher
from ThermoFisher, BioMek Systems from
Beckman Coulter and the Tecan Genesis from
Tecan AG.
Biotinylated proteins and oligonucleotides, in
association with streptavidin-coated magnetic
particles, are being used in clinical diagnostic
assays, proteomic and genomic assays
including:
- Immunoassays/Immunodiagnostics
- Purification of DNA/RNA binding
- proteins
- Protein purification
- Biopanning
- Cell isolation
- Gene sequencing
Optimizing the Assay
Assay developers are faced with two over-riding
considerations when developing a streptavidin
magnetic bead based assay; optimizing the
assay for the highest signal and for the lowest
non-specific binding (NSB). For optimization it
is important to consider biotin-binding capacity.
How can these assays be optimized?
Increase the biotin-binding capacity:
Magnetic beads produce non- specific binding
and therefore background noise. By increasing
binding, the signal on each bead can be greatly
increased. As biotin-binding capacity increases
less beads are required resulting in less
background from the use of less beads. Higher
capacity leads to reduced background and a
more sensitive assay.
Increase the surface area of the beads:
Small sized beads have greater surface area
resulting in increased loading of streptavidin
and therefore will lead to higher signal per
bead. Many sizes of bead exist, the most
popular size being 2.8µm. Therefore, a bead
that has a smaller diameter will allow for much
better signal.
Indirect detection or capture assays do not
require uniformed sized beads: Assays which
employ enzyme-mediated signal generation,
like ELISA, require high biotin binding capacity
but not uniformed sized beads. For these types
of assays it is most important that biotin-
binding/mg is consistent lot-to-lot for assay
reproducibility. Uniform particles are only
necessary when the signal is detected directly on the bead by visual means (i.e., single
molecule detection).
Current High Capacity Streptavidin Beads on
the market
There are many magnetic streptavidin bead
manufacturers, and the beads can all use the
same magnetic platforms because at their core,
they are all made from the iron magnetite
material. The beads differ in their size,
uniformity, magnetic response time and most
import, in their capacity to bind biomolecules.
By covalently crosslinking streptavidin using a
proprietary conjugation chemistry, Solulink is
able to efficiently increase streptavidin loading
with high stability and low streptavidin leaching,
as shown in Figure 1. This chemistry produces
the highest biotin binding capacity of any bead
on the market; greater than 12 nmol biotin/mg
of bead.
Figure 2 compares biotin-binding capacity of
Solulink’s NanoLink Streptavidin beads to the
published data of other commercially available
streptavidin-magnetic beads.
As described, assays have varying requirements
for streptavidin-magnetic beads. NanoLink
Streptavidin beads are polydispersed of average
diameter of 800 nm and possess biotin binding
capacity of >12 nmol/mg. While the beads can
be used for a variety of applications, they are
best suited for protein capture using
biotinylated DNA, peptides or other proteins as
bait.
Ultra-high capacity microspheres reduce the
overall particle mass required to immobilize
biotinylated proteins and oligonucleotides. This
leads to reduced costs and lower non-specific
background (NSB).
Demonstration of the low background binding
and high biotinylated-antibody capture
efficiency of the NanoLink beads is presented in
Figure 3. The overlaid UV spectra on the left
shows minimal non specific binding of bovine
IgG to NanoLink beads. The graph on the right
presents the binding of biotinylated bIgG (using
Solulink’s ChromaLink Biotin Reagent) to
NanoLink beads.
Conclusion
Solulink’s NanoLink Streptavidin Magnetic
Beads offer unparalled binding capacity
allowing scientists to optimize the efficiency of
their magnetic assays.
Solulink offers the following resources to successfully use NanoLink Streptavidin
Magnetic Beads and ChromaLink Biotin Labeling reagent:
NanoLink Protocol:
Link
ChromaLink Biotin Protocol:
Link
Solulink Catalog Listings
