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- [email protected]
Cat. No.
Product Name
Unit Size
Order
Specification
Composition
Magnetic beads are grafted with a Sulfhydryl group on the surface
Number of Beads
~ 1.68 x 109 beads/mg (1μm beads)
~ 5 x 107 beads /mg (5μm beads)
Magnetization
~40-45 EMU/g
Type of Magnetization
Superparamagnetic
Effective Density
2.5 g/ml
Formulation
Lyophilized Powder
Functional Group Density
1μm Magnetic Beads
~250 μmole / g of Beads
5μm Magnetic Beads
~200 μmole / g of Beads
1μm Long-Arm Magnetic Beads
~220 μmole / g of Beads
5μm Long-Arm Magnetic Beads
~185 μmole / g of Beads
Storage
Upon receipt, store at 4°C
In chemistry, a sulfhydryl group, also known as a thiol, is a functional group containing a sulfur atom linked to a hydrogen atom. It is often denoted by the suffix “-thiol” and the prefix “mercapto-” or “sulfanyl.” These groups have a strong affinity for soft metals. BcMag™ has developed Sulfhydryl-Terminated Magnetic Beads that are uniform, silica-based superparamagnetic beads grafted with a high density of sulfhydryl functional groups on the surface. These beads are specifically designed for conjugating protein/peptides modified by SMCC, which contains an amine-reactive N-hydroxysuccinimide and a sulfhydryl-reactive maleimide group. The conjugation procedure is quick and easy, eliminating the need for laborious pipetting and centrifuging. BcMagTM Sulfhydryl-Terminated Magnetic Beads are best suited for conjugating large proteins, while BcMagTM Long-Arm Sulfhydryl-Terminated Magnetic Beads are recommended for small peptides.
Protocol
Note:
●
This protocol can be scaled up as needed. We strongly recommended titration to optimize the number of beads used for each application.
Materials Required
●
Conjugation buffer: PBS, PH 7.2
●
SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate ( Pierce, Cat. No. 22360 22122) or Sulfo-SMCC (Pierce, Cat. No. 22122)
●
Magnetic Rack (for manual operation)
Based on sample volume, the user can choose one of the following Magnetic Racks:
1. BcMag™ Magnetic Rack-2 for holding two individual 1.5 ml centrifuge tubes (Cat. No. MS-01);
2. BcMag™ Magnetic Rack-6 for holding six individual 1.5 ml centrifuge tubes (Cat. No. MS-02);
3. BcMag™ Magnetic Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Cat. No. MS-03);
4. 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);
5. BcMag™ Magnetic Rack-96 for holding a 96 ELISA plate or PCR plate (Cat. No. MS-05).
Protein /Peptide Preparation
1.
Dialyse the protein/Peptides against 50 volumes of PBS, pH 7.2, 5mM EDTA.
2.
Add the appropriate amount of SMCC to the protein solution and mix very well.
Notes: Prepare SMCC solution (3.7mg/ml in DMF. If the SMCC does not completely dissolve, place the tube in a 50°C water bath for several minutes.) Add 100 μl of SMCC solution to 1 ml protein solution (10mg/ml), or 50 μl of SMCC solution to 1 ml protein solution (<1mg/ml).
3.
Incubate at + 4°C for 2 hours or room temperature for 30 minutes.
4.
Remove free SMCC and unmodified proteins on a Sephadex G15 column. Elute with PBS buffer. The elution of the protein-SMCC can be monitored at 206 nm with a spectrophotometer.
Magnetic Beads Preparation
Note: Weight, suspend the magnetic beads with PBS (Concentration: 30mg/ml), disperse the beads by vigorous vortexing, and store at 4˚C. Shake the bottle to resuspend the Magnetic Beads before use completely.
1.
Shake the bottle to resuspend the BcMag™ Sulfhydryl-Terminated- Magnetic Beads thoroughly.
2.
Transfer 100μl-300 μl of the Beads (30 mg/ml) to a tube. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
3.
Remove the tube and resuspend the beads thoroughly with 200μl PBS buffer. Leave the tube at room temperature for 2-3 minutes. Place the tube on the magnetic rack for 1-3 minutes. Remove the supernatant while the tube remains on the rack.
4.
Repeat step 3 two times.
5.
Incubate the Magnetic Beads with 1 ml of DTT (dithiothreitol 3 mg/ml) for 15 minutes at room temperature and wash the Beads three times with PBS buffer.
6.
Mix the reduced beads with the protein-SMCC conjugate, and incubate for 12 hours at +4°C.
7.
Wash the beads and resuspend them in the desired buffer.
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