Protein A MagBeads
Cat. No. L00273
Technical Manual No. TM0248
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Version 04202010
Description ….……………………………………………………………………………..….
Key Features ….……………………………………………………………………. …….….
Characteristics…………………………… …………………………………………………
General Protocols…………………………..…………………………………………………
Example ………………………………………………………………………………………..
Troubleshooting .………………….…………………………………………………………
Ordering Information…………………………………………………………………..……...
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I. DESCRIPTION
Protein A MagBeads(Cat. No. L00273, 4 ml 25% slurry) are average 40µm in size, superparamagnetic beads coated
with recombinant protein A. The Protein A MagBeads are suitable for use in binding immunoglobulins, as an effective
adsorbant for removal of Fc fragments during Fab fragment preparation, in immunoassay systems, and they are
ready to use after one quick vortex or even a vigorous shake. The beads are supplied as 25% slurry in phosphate
buffered saline (PBS, pH 7.4) containing 20% ethanol.
Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus, binds to mammalian IgGs, mainly
through Fc regions. Native Protein A has five IgG binding domains and many unknown-function repeated sequences.
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Recombinant Protein A contains five high-affinity (Ka=10 /M) IgG binding domains to reduce nonspecific binding.
II. KEY FEATURES
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Quick and convenient separation accomplished by magnetic means
High capacity: The product can manage over 10 mg/ml of porcine IgG
Low rate of nonspecific binding
Optimized homogeneous recombinant ligands
III CHARACTERISTICS
Recombinant Streptococcal protein A expressed in E.
coli
5
≈ 35 kDa
5.17
≈ 2 mg protein A/ml settled beads
>10 mg porcine IgG/ml settled beads
Bio-magnetic agarose, 4%
20-75 μm
2-8°C
12 months at 2-8 ℃
Ligand
Number of IgG binding sites per ligand
Molecular weight of ligand
pI of ligand
Concentration of ligand
Static binding capacity
Material
Particle size
Storage
Shelf life
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IV. GENERAL PROTOCOL
A. Additional materials required
Binding buffer: Na2HPO4 20 mM, NaCl 0.15 M, pH 7.0
Elution buffer: 0.1 M glycine, pH 2-3
Neutralization buffer: 1 M Tris; pH 7.5-9
1.5 ml microcentrifuge tubes
Magnet for a 1.5 ml microcentrifuge tube
B. Procedure
NOTE:
Shake beads vigorously before use
1) Place 100 μl of the BioMag Beads into a 1.5 ml microcentrifuge tube.
2) Add 1 ml of binding buffer to the tube and invert tube several times to mix. Use the magnet to separate the
beads. Once the supernatant becomes clear, remove and discard it. Repeat this step three more times.
3) Resuspend the beads in 100 μl of binding buffer.
4) Add 50 μl of serum or cell culture supernatant to the tube and gently invert tube to mix. Incubate tube at room
temperature with mixing for one hour.
5) Magnetically separate the beads. Once the supernatant becomes clear, remove and discard the supernatant.
6) Add 1000 ml of binding buffer to the tube, mix well, magnetically separate the beads and discard it. Repeat this
wash three more times.
7) Add 100 μl of elution buffer to the tube. Mix well and incubate for five minutes at room temperature with
occasional mixing. Magnetically separate the beads. Once the supernatant becomes clear, remove and
reserve it. It contains the eluted antibody. Repeat this elution three more times to recover as much of the IgG
as possible.
8) To neutralize the low pH, add 2.5 μl of neutralization buffer for each 50 μl of eluate. If desired, perform a buffer
exchange by dialysis or desalting.
V. EXAMPLES
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Fig.1. SDS-PAGE of binding capacity detection of Protein A
MagBeads (GenScript, L00273)
1. Flow 2. Wash 3. Elute 4. Molecular standard
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V I. TROUBLESHOOTING
Problem
The magnetic particles are hard to
immobilize with the magnet.
Possible Cause
There are too many Protein A
MagBeads for the magnet to
properly immobilize them all.
Solution
Decrease the volume of MagBeads
suspension.
A considerable amount of antibody
has been purified, but
no specific antibody of interest is
detected.
The antibody of interest is at very
low concentration.
Use a serum-free medium for cell
supernatant samples.
The antibody of interest is purified,
but it is
degraded (as determined by lack of
function in downstream assay).
The antibody is sensitive to low-pH
elution buffer.
No antibody is detected in any
elution
Fraction.
The sample devoid of any antibody
species or subclass that binds to
Protein A.
Affinity-purify the antibody using its
specific antigen coupled to an
affinity support.
The downstream application is
sensitive to the neutralized elution
buffer.
Try another elution reagent, such as
3.5 M MgCl2 , 10 mM phosphate, pH
7.2.
Desalt or dialyze the eluted sample
into a
suitable buffer.
Try GenScript Protein G Resin
MagBeads.
VII. ORDERING INFORMATION
GenScript Protein A MagBeads, Cat. No. L00273
For Research Use Only.
GenScript USA Inc.
120 Centennial Ave., Piscataway, NJ 08854
Tel: 732-885-9188, 732-885-9688
Fax: 732-210-0262, 732-885-5878
Email: [email protected]
Web: www.genscript.com
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