- +1 858 909 0079
- +1 858 909 0057
- [email protected]
- +1 858 909 0079
- [email protected]
Specification
Composition
Magnetic beads grafted with C18 alkyl groups
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
Formulation
Lyophilized Powder
Binding Capacity
1 μm beads: >20 μg protein/mg of Beads
5 μm beads: >18 μg protein/mg of Beads
Storage
Store at 4°C upon receipt.
Hydrophobic magnetic resins offer a uniform and stable alternative to traditional chromatographic techniques for protein purification. These rigid polymeric beads have covalent surface chemistries and are grafted with a high density of hydrophobic ligands, making them easy to handle and pack. They are manufactured using superparamagnetic iron oxide as the core, which is encapsulated by a high-purity silica shell to prevent leaching. This pure, inert silica minimizes nonspecific binding and results in higher resolution due to the smaller size and non-porous nature of the beads. By replacing time-consuming and expensive chromatography methods, hydrophobic magnetic resins provide a robust solution for protein purification.
BcMag™ C18 Magnetic Beads are a uniform and superparamagnetic option for purifying, desalting, and concentrating peptides and proteins in the femtomolar to picomolar range. With hydrophobic C18alkyl groups on their surface, these beads can replace laborious pipetting and centrifugation procedures, and can be used manually or automatically. While recommended for low molecular weight proteins or peptides, BcMag™ C18 magnetic beads can be interchanged with BcMag™ C-8 and C-4 magnetic beads, depending on the molecular weight of the protein or peptide. For low to intermediate molecular weight proteins, BcMag™ C-8 magnetic beads are preferred, while BcMag™ C-4 magnetic beads are most suitable for larger molecular weight proteins and peptides.
Note:
●
To achieve maximum binding to the BcMag™ C18Magnetic Beads, TFA (trifluoroacetic acid) or other ion-pairing agents should be between 0.1%–1.0% at a pH of <4. The solvents should be completely removed if samples contain excess organic solvents such as methanol or acetonitrile (ACN). Samples can be dried in a vacuum evaporator and resuspended in sample buffer (below). To optimize binding, detergents in samples should be diluted with 0.1% TFA till SDS <0.1%, or Triton ® <1%, or Tween® <0.5%.
●
To avoid excessive beads drying between steps, the entire procedure should be carried out in a timely manner.
●
The amount of beads used in each application should be empirically titrated. The volumes can be scaled up or down accordingly. We recommend using 10 μl (0.5 mg) BcMag™ C18Beads for binding of ~ 10 μg protein and 5μl elution buffer for 0.5 mg beads.
●
To get the best results, users are encouraged to determine the optimal working conditions based on the protocol and suggestions described in the Troubleshooting section.
Materials Required
●
Buffers
Equilibration buffer: 0.5% TFA (trifluoroacetic acid) in 5% ACN (acetonitrile)
Sample Binding Buffer: 2% TFA in 5% ACN
Washing buffer: 0.5 % TFA in 5% ACN
Elution Buffer: 70% ACN
●
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).
Procedure
A.
Magnetic Beads Preparation
1.
Weight and suspend 50 mg beads with 1ml of 50% methanol.
2.
Transfer 10μl (50 mg/ml) of completely suspended magnetic beads to a microcentrifuge tube.
3.
Place the tube onto a magnetic separator for 1-3 minutes until the supernatant is clear.
4.
Wash the beads with 1 ml of Equilibration buffer by a magnetic separator.
5.
Aspirate and discard the supernatant with a pipette while the tube remains in the separator.
B.
Sample Binding
1.
Mix sample (~10μg protein/ peptide) with 1/3 volume of Sample Binding Buffer and add to the tube containing the beads.
2.
Thoroughly mix beads and sample using a pipette and leave at room temperature for 2 minutes to allow proteins to bind to the beads.
3.
Place the tube onto the magnetic separator for 1-3 minutes (no longer than 3 minutes) until the supernatant is clear. Aspirate and discard the supernatant with a pipette while the tube remains in the separator.
4.
Remove the tube from the separator and resuspend the beads with 100μl washing buffer.
5.
Place the tube onto the magnetic separator for 1-3 minutes until the supernatant is clear. Aspirate and discard the supernatant with a pipette while the tube remains in the separator.
6.
Repeat steps 2 to 4 for four times.
C.
Elution
1.
Remove the tube from the separator, add 5μl elution buffer, resuspend the beads and incubate for 2 minutes at room temperature.
2.
Place the tube on the magnetic separator for 1-3 minutes and transfer the supernatant containing the eluted protein to a new tube. (User should optimize elution conditions for individual proteins by adjusting acetonitrile concentrations, such as 20%, 50%, 80%).
3.
For MALDI-MS analysis, mix 1μl of the eluate with 1μl of matrix solution and spot 0.5μl onto a MALADI-MS target plate.
Troubleshooting
Problem
Poor absorption of proteins/Peptides to beads.
Probable Cause
Hydrophobic interaction is not strong enough.
Suggestion
Increase the NaCl concentration (up to 0.2 M) used during adsorption.
Problem
Poor absorption of proteins/Peptides to beads.
Probable Cause
Biomolecules are not completely solubilized in the sample buffer.
Suggestion
Use denaturing conditions during adsorption. Add Guanidine HCl to the sample to achieve a final concentration between 1– 6 M.
Problem
Poor absorption of proteins/Peptides to beads.
Probable Cause
The sample’s chemical properties do not support hydrophobic interaction with reverse-phase beads.
Suggestion
Choose suitable reverse phase beads for your sample.
Problem
Poor elution from BcMag™ C18 Beads
Probable Cause
Hydrophobic interaction is too strong.
Suggestion
Increase the acetonitrile concentration used during elution. Decrease the NaCl concentration used during adsorption.
Problem
Poor elution from BcMag™ C18 Beads
Probable Cause
Proteins/peptides are not readily soluble in organic solutions.
Suggestion
Decrease the organic solvent concentration used during elution.
Problem
Poor elution from BcMag™ C18 Beads
Probable Cause
Protein-bound too tightly to beads
Suggestion
Choose more suitable reverse phase beads for your sample.
Problem
Poor yield
Probable Cause
The quantity of protein or peptides of interest in the sample is too low.
Suggestion
Problem
Probable Cause
Suggestions
Poor absorption of proteins/Peptides to beads.
Hydrophobic interaction is not strong enough.
Increase the NaCl concentration (up to 0.2 M) used during adsorption.
Biomolecules are not completely solubilized in the sample buffer.
Use denaturing conditions during adsorption. Add Guanidine HCl to the sample to achieve a final concentration between 1– 6 M.
The sample’s chemical properties do not support hydrophobic interaction with reverse-phase beads.
Choose suitable reverse phase beads for your sample.
Poor elution from BcMag™ C18 Beads
Hydrophobic interaction is too strong.
Increase the acetonitrile concentration used during elution. Decrease the NaCl concentration used during adsorption.
Proteins/peptides are not readily soluble in organic solutions.
Decrease the organic solvent concentration used during elution.
Protein-bound too tightly to beads
Choose more suitable reverse phase beads for your sample.
Poor yield
The quantity of protein or peptides of interest in the sample is too low.
1.
Lauber MA, Koza SM, McCall SA, Alden BA, Araneta PC, Fountain KJ. High-resolution peptide mapping separations with MS-friendly mobile phases and charge-surface-modified C18. Anal Chem. 2013 Jul 16;85(14):6936-44.
2.
Field JK, Euerby MR, Haselmann KF, Petersson P. Investigation into reversed-phase chromatography peptide separation systems Part IV: Characterisation of mobile phase selectivity differences. J Chromatogr A. 2021 Mar 29;1641:461986.
3.
Liang Y, Wang C, Liang Z, Zhang L, Zhang Y. C18-Functionalized Amine-Bridged Hybrid Monoliths for Mass Spectrometry-Friendly Peptide Separation and Highly Sensitive Proteomic Analysis. Anal Chem. 2022 Apr 26;94(16):6084-6088.
4.
Wang Q, Ye M, Xu L, Shi ZG. A reversed-phase/hydrophilic interaction mixed-mode C18-Diol stationary phase for multiple applications. Anal Chim Acta. 2015 Aug 12;888:182-90.
5.
Hernandez-Hernandez O, Quintanilla-Lopez JE, Lebron-Aguilar R, Sanz ML, Moreno FJ. Characterization of post-translationally modified peptides by hydrophilic interaction and reverse-phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. J Chromatogr A. 2016 Jan 8;1428:202-11.
6.
Zhou, N. E., Mant, C. T., Kirkland J. J., and Hodges R. S. (1991) Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins. J. Chromatogr.548, 179–193.
Get the Latest News and Updates by Email
6393 Nancy Ridge Dr. Suite A
San Diego, CA 92121 USA
Fax: +1-858-909-0057
Get the Latest News and Updates by Email
© 2023 Bioclone Inc. All Rights Reserved.
Magnetic Beads Make Things Simple
