- +1 858 909 0079
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- +1 858 909 0079
- [email protected]
Specification
Composition
Magnetic beads grafted with the weak cation-exchange group.
Number of Beads
~ 1.68 x 109 beads/mg (1μm beads)
~ 5 x 107 beads /mg (5μm beads)
Magnetization
~45 EMU/g
Type of Magnetization
Superparamagnetic
Effective Density
2.0 g/ml
Stability
Most organic solvents
Weak Cation Exchange Beads
1.0 μm beads: >2.5 mg Lysozyme / ml of Beads
5 μm beads: >2 mg Lysozyme / mlof Beads
Storage
Store at 4°C upon receipt.
The utilization of magnetic beads in the field of molecular biology has become an indispensable tool for the fractionation and purification of proteins and nucleic acids from complex biological samples. Magnetic beads employ adsorbent technology, acting as a chromatographic matrix, to fractionate the target molecule from raw biological materials. The ion exchange chromatography process is commonly used for this purpose, taking advantage of differences in surface charges to separate molecules of interest.
BcMag™ Weak Cation Exchange (WCX) Magnetic Beads are a specific type of magnetic bead that possess uniformity in size and shape, with a high density of carboxylate functional groups grafted onto the surface. These magnetic beads are functionalized with a weak cation exchanger resin, allowing for the rapid release of strong ions that may be retained irreversibly on Strong Ion Cation (SCX) beads. Moreover, weak ion exchangers can also be used as effective separation tools when strong ion exchangers fail, given the differences in selectivity between the two.
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Fast and simple – WCX Magnetic resins-based format eliminates columns or filters or a laborious repeat of pipetting or centrifugation.
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Convenient and expandable – WCX Magnetic format enables high-throughput processing of multiple samples in parallel with many different automated liquid handling systems.
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Robust – Magnetic beads do not crack or run dry.
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Low bed volume – Working with small magnetic bead volumes allows for minimal buffer volumes, resulting in concentrated elution fractions.
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Protein pre-fractionation in cell lysates
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Optimizing purification conditions for new protein preparation protocols
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Protein purification and concentration
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Antibody purification from serum, ascites, or tissue culture supernatant
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Preparation of samples before 1D or 2D PAGE
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Phosphopeptide purification before MS analysis
Note: The following protocol is an example of fractionating a protein or peptide sample with BcMag™ WCX magnetic beads. Users are encouraged to choose alternative binding, washing, or elution buffers to get the best results and determine the optimal working conditions based on the protocol and suggestions described in the troubleshooting section. It is critical to match the amount of the beads to the amount of protein in the starting material in all protein purification experiments. It is not only for financial reasons but also because insufficient WCX resin results in inadequate protein binding in the solution. Too many affinity binding sites will result in the binding of other proteins, making the purification less selective and requiring extra purification steps to achieve pure protein. We recommend performing a titration to optimize the beads used for each application. It is necessary to optimize volumes of elution to avoid unnecessary sample dilution.
Note: Select the appropriate buffer
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Based on the protein’s pI, empirically calculate the appropriate buffer (pH and salt concentration) for purifying and eluting the protein of interest. In a buffered solution above the protein’s pI, the protein becomes negatively charged (deprotonated) and binds to the positively charged functional groups of an anion exchange resin. To choose the correct buffer for a selected pH, the following is a general rule for selecting a buffer pH:
Anion exchanger — 0.5–1.5 pH units higher than the protein’s pI of interest.
Cation exchanger — 0.5–1.5 pH units lower than the protein’s pI of interest.
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Binding/Wash Buffer: The pKa of the BcMag™ weak cation exchange magnetic beads is about 5.0. The pH of Binding/Wash Buffer should be 7.0 or above and at least one pH unit away from the pI of the target protein or peptide. At pH 7.0 or above, the beads carry a negative charge.
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Elution Buffer: To elute the target protein or peptide from the magnetic beads, user should optimize elution condition for individual application by stepwise elution either using solutions with increasing salt concentration (50 mM sodium phosphate pH 7.0, 50 mM NaCl, followed by 50 mM sodium phosphate pH 7.0 100 mM NaCl, then 50 mM sodium phosphate pH 7.0, 200mM NaCl, then 50 mM sodium phosphate pH 7.0, 400 mM NaCl, then 50 mM sodium phosphate pH 7.0, 800 mM NaCl, and 50 mM sodium phosphate pH 7.0, 1.0 M NaCl), or using 0.5% trifluoroacetic acid (TFA) in water.
A. Equipment
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Magnetic Rack (for manual operation)
Based on sample volume, the user can choose one of the following Magnetic Racks:
– BcMag™ Magnetic Rack-2 for holding two individual 1.5 ml centrifuge tubes (Cat. No. MS-01);
– BcMag™ Magnetic Rack-6 for holding six individual 1.5 ml centrifuge tubes (Cat. No. MS-02);
– BcMag™ Magnetic Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Cat. No. MS-03);
– BcMag™ Magnetic Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Cat. No. MS-04);
– BcMag™ Magnetic Rack-96 for holding a 96 ELISA plate or PCR plate (Cat. No. MS-05).
For larger scale purification, Ceramic Magnets Block for large scale purification (6 in x 4 in x 1 in block ferrite magnet, Applied Magnets, Cat. No. CERAMIC-B8).
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Corning 430825 cell culture flask for large-scale purification (Cole-Parmer, Cat. No. EW-01936-22)
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Mini BlotBoy 3D Rocker, fixed speed, small 10″ x 7.5″ platform w/ flat mat (Benchmark Scientific, Inc. Cat. No. B3D1008) or compatible
B. Buffer
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Precondition Buffer: 50 mM Sodium phosphate, pH 7.
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Binding/Wash Buffer: 50 mM Sodium phosphate, pH 7.0, 20 mM NaCl
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Elution Buffer: 50 mM Sodium phosphate, pH 7.0, 0.1-1.0 M NaCl
General Protocol for using the Weak Anion Exchange Magnetic Beads –
a.
Weak cation exchange magnetic beads preparation
1.
Vigorously shake the bottle until the magnetic beads become homogeneous and transfer an appropriate volume of the magnetic beads from the bottle to a new tube or flask.
Note:
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Optimize the number of beads used for each application. Too many beads will cause higher background. Insufficient beads will lead to lower yields.
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2.
3.
Repeat step (2) one more time.
4.
Equilibrate the beads by adding ten bead-bed volumes of Binding/Washing buffer and shake it to mix them. Incubate at room temperature with continuous rotation for 2 minutes. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack. The beads are ready for purification.
b.
Purification
1.
Add the equilibrated beads (Step a (4)) to the sample and incubate on Mini BlotBoy 3D Rocker with continuous rotation for 5-10 minutes.
2.
Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack. Add ten bead-bed volumes of Binding/Washing buffer and shake it ten times to wash the beads. Again, place the tube on the magnetic rack for 1-3 minutes and remove the supernatant while the tube remains on the rack.
3.
Repeat step (2) six times.
Note:
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This step is critical to get high pure protein. It may be necessary to wash the beads more than six times for some proteins to reduce the nonspecific binding.
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Optimize the washing buffer (pH and salt concentration)
Elute protein with an appropriate volume of elution buffer by pipetting up and down 10-15 times or vortex mixer for 5 minutes.
Note:
Determine the optimum elution buffers (pH and salt concentration) and eluting the protein 2-3 times may be necessary.
4.
Elute protein with an appropriate volume of elution buffer by pipetting up and down 10-15 times or vortex mixer for 5 minutes.
Note:
Determine the optimum elution buffers (pH and salt concentration) and eluting the protein 2-3 times may be necessary.
5.
Collect and transfer the supernatant to a new tube.
C. Troubleshooting
Problem
Low yield
Probable Cause
The sample’s ionic strength is high.
Suggestion
Problem
Low yield
Probable Cause
The sample contains interfering detergents.
Suggestion
Problem
The protein failed to elute.
Probable Cause
Ionic interaction is too strong.
Suggestion
Problem
Poor separation
Probable Cause
Carry-over between eluted fractions
Suggestion
Problem
Poor separation
Probable Cause
Proteins or peptides with similar pI to the target protein
Suggestion
Problem
Probable Cause
Suggestions
Low yield
The sample’s ionic strength is high.
The sample contains interfering detergents.
The protein failed to elute.
Ionic interaction is too strong.
Poor separation
Carry-over between eluted fractions
Proteins or peptides with similar pI to the target protein
1.
Wittkopp F, Peeck L, Hafner M, Frech C. Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model. J Chromatogr A. 2018 Apr 13;1545:32-47.
2.
Staby A, Jacobsen JH, Hansen RG, Bruus UK, Jensen IH. Comparison of chromatographic ion-exchange resins V. Strong and weak cation-exchange resins. J Chromatogr A. 2006 Jun 23;1118(2):168-79.
3.
Ludewig R, Nietzsche S, Scriba GK. A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC. J Sep Sci. 2011 Jan;34(1):64-9.
4.
Chen X, Tolley HD, Lee ML. Weak cation-exchange monolithic column for capillary liquid chromatography of peptides and proteins. J Sep Sci. 2011 Aug;34(16-17):2063-71.
5.
Pabst TM, Antos D, Carta G, Ramasubramanyan N, Hunter AK. Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups. J Chromatogr A. 2008 Feb 15;1181(1-2):83-94.
6.
Xu W, Regnier FE. Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations. J Chromatogr A. 1998 Dec 18;828(1-2):357-64.
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