Magnetic beads are useful to purify proteins from dilute solutions, or when only low expression levels are achieved. PureCube Glutathione MagBeads are ferrimagnetic agarose beads coupled to glutathione, an efficient ligand for GST fusion proteins. PureCube MagBeads are produced in a very robust, reproducible way to minimize lot-to-lot variability. Our MagBeads are homogeneous in size, and provide the identical surface chemistry as PureCube Glutathione Agarose
, making magnetic beads an excellent tool for fast screening of binding conditions. PureCube Glutathione MagBeads are provided as a 25% suspension.
PureCube Glutathione MagBeads provide:
- Binding capacity of up to 12 mg protein per ml settled beads
- purification of proteins expressed at low levels or from diluted solutions
- optimal conditions for pull-down experiments
- low unspecific binding
- Also available as Glutathione Agarose and prepacked cartridges
PureCube Gluathione MagBead's efficiency
Fig.1: Reliable lot-to-lot reproducibility. Glutathione-S-transferase was purified from E.coli lysates using three independent production lots of PureCube Glutathione MagBeads. FT: Flow through, E1-E5: Elution fractions.
GST/Gluthathione MagBeads from Cube Biotech was successfully used in the following publications and patents:
||Specific binding and purification of GST-tagged proteins
||Affinity to GST-tagged proteins
|High binding capacity
||10 mg/mL settled beads
||Delivered as a 25 % suspension
|A.||Protein purification protocol:|
- Thaw the E. coli cell pellet on ice. Optional: Freezing the cell pellet at -20 °C for 30 min prior to incubation at room temperature improves lysis by lysozyme.
- Resuspend the cell pellet in 1 mL Lysis Buffer. Add 30 U Benzonase® (3 units/mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA.
- Incubate for 30 min on ice, if necessary for protein stability. Otherwise, incubating at room temperature (20-25 °C) may be more efficient.
- Centrifuge the lysate for 30 min at 10,000xg and 2-8 °C. Collect the supernatant. Note: The supernatant contains the cleared lysate fraction. We recommend to take aliquots of all fractions for SDS-PAGE analysis.
- Resuspend the PureCube Glutathione MagBeads by vortexing. Transfer 40 μL of the 25 % magnetic beads suspension into a conical microcentrifuge tube. Note: Depending on the protein expression rate, the quantity of magnetic bead suspension can be adjusted from 2-200 μL.
- Add 500 μL Lysis Buffer to the Glutathione MagBeads and mix by vortexing. Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant.
- Pipet 1 mL of the cleared lysate onto the equilibrated MagBeads, and incubate the mixture at 4 °C for 1 h on an end-over-end shaker.
- Place the tube on the magnetic microtube stand until the beads separate and remove the supernatant.
- Remove the tube from the magnet. Add 500 μL Wash Buffer and mix by vortexing. Place the tube again on the magnetic microtube stand and allow the beads to separate. Remove the supernatant.
- Repeat step 10 twice. 12: Optional: To remove contaminants such as chaperones, perform an additional wash step with ATP buffer.
- Elute the GST-tagged protein using 100 μL Elution buffer. Note: Depending on the protein expression rate and desired protein concentration, the elution volume can be adjusted from 25 to 500 μL.
- Repeat step 12xxx Collect each elution fraction in a separate tube.
- Analyze all fractions by SDS-PAGE. Note: Do not boil membrane proteins. Instead, incubate the sample at 46 °C for 30 min in preparation for SDS-PAGE analysis.
- Optional: Perform Western Blot experiment using GST Antibody.
1. Chiocchini C., Trefzer A., Vattem K., Kuhn P., inventor; Thermo Fisher Scientific Geneart GmBH, Pierce Biotechnology Inc, assignee. Devices and methods for producing nucleic acids and proteins. United States patent US20180291413A1. 2016 April 10.