M-Beads Magnetic Silica Beads WAX

Transcription

1 M-Beads Magnetic Silica Beads WAX

2 MoBiTec GmbH 2012 Page 2 Contents Technical data... 3 Application... 4 General information... 4 Bead usage... 4 Additional materials needed... 4 Protocols... 5 Order Information, Shipping and Storage... 7 Contact and Support... 7

3 MoBiTec GmbH 2012 Page 3 Technical data Product Name Mean Size Concentration Supplied product volume Material M-Beads Magnetic silica beads WAX 1.2 μm (+/-0.1 µm) 20 mg/ml 2 ml, 10 ml Magnetic silica beads with high magnetic content and low sedimentation. Sedimentation Solution additives Filtered demineralized water Storage Store at 2-8 C Material supplied Vial with silica beads suspended in filtered demineralized water.

4 MoBiTec GmbH 2012 Page 4 Application General information M-Beads Magnetic silica beads WAX (weak anion exchange) are an ideal tool for the reduction of protein or peptide complexity e.g. cell lysates. Main applications include: sample preparation and pre-fractionation prior to mass spectrometry (e.g. MALDI-TOF analysis) SDS-PAGE analysis protein and peptide separation for multiple downstream applications biomarker analysis and serum/plasma profiling M-Beads Magnetic silica beads WAX are magnetic silica beads with a high magnetic content optimized for protein and peptide separation. The high magnetic strength makes them applicable for both, manual and automated/robotic fractionation, because the beads will typically collect in less than 1 minute when magnetic force is applied. This quick and complete separation gives very good reproducibility since no beads will be lost during washing steps. Furtheron the quick protein adsorption, desorption and magnetic collection typically shortens significantly the protocol time over conventional column based ion exchange chromatography. Bead Usage This product is stable for at least 1 year after production date when stored at 2-8 C. Store beads in well closed vial and in upright position to prevent drying of the beads since this makes them more difficult to resuspend. Do not freeze the product! Vortex bead suspension well before use. The beads are suspended in filtered water. This suspension media can easily be replaced with your own buffer/storage media. The beads can be used in a ph range from 3 to 12. At these conditions, no degradation or iron oxide leakage was measurable using spectrophotometric assays. The beads are proved to be compatible with mass spectrometry workflows. Nevertheless, if you expect iron interference in downstream applications, we strongly advise you to rinse the beads before use. Additional Materials Needed Depending on the application, some buffers and materials are needed. Magnets for bead separation/collecting. Own suspension buffers. Lysis, binding, washing and elution buffer. Mixer/vortex to mix samples and resuspend beads

5 MoBiTec GmbH 2012 Page 5 Protocols 1) Buffers and reagents needed: M-Beads Magnetic silica beads WAX (conc. 20 mg/ml) Preload solution: Load WCX beads with counter ions: 0.02 M bis Tris ph 6 plus 1 M NaCl Adsorption solution: 0.02 M bis Tris, ph 6 Washing solution: water (HPLC grade) Desorption solution 1 for Maldi MS: 1% TFA in water Desorption solution 2: a) 0.02 M bis Tris, ph 6, 0.05 M NaCl b) 0.02 M bis Tris, ph 6, 0.1 M NaCl c) 0.02 M bis Tris, ph 6, 0.15 M NaCl d) 0.02 M bis Tris, ph 6, 0.20 M NaCl e) 0.02 M bis Tris, ph 6, 0.25 M NaCl Optional adsorption and desorption buffers: For efficient adsorption and desorption the ph of the adsorption and desorption buffer should be at least one ph unit above the pi of the molecule to be bound. On the other hand the difference between adsorption/desorption buffer ph and pi of the target molecule should not differ over several ph units. Therefore further buffer systems should be taken into account depending on the pi of your target molecule. For analyzing body fluids like serum and others we recommend to test further buffer systems at different ph anyhow, since typically the pi of the target molecule(s) is unknown: 0.1 % TFA (ph < 3.0) sodium citrate buffer, ph N-Methylpiperazine, 20 mm, ph Piperazine, 20 mm, ph bis Tris, 20 mm, ph bis Tris propane, 20 mm, ph Triethanolamine, 20 mm, ph For the corresponding desortption solution (salt step gradient) 0.05 M, 0.1 M, 0.15 M, 0.2 M and 0.25 M NaCl has to be added like for the Sodium phosphate buffer above. Optional use of detergents For the corresponding desorption solution (salt step gradient) 0.05 M, 0.1 M, 0.15 M, 0.2 M and 0.25 M NaCl has to be added like for the Tris buffer above.

6 MoBiTec GmbH 2012 Page 6 2) Counter ions preload 1. Vortex M-Beads Magnetic silica beads WAX to a homogeneous suspension. 2. Transfer 20 µl slurry to an PCR tube. 3. Place the tube to the magnet for 1-2 minutes. 4. Remove the supernatant. 5. Remove the tube from the magnet. 6. Add 200 µl preload solution and resuspend. 7. Magnetic separation for 2 minutes, discard the supernatant. 8. Repeat step 6. and 7. two times. 3) Equilibration to adsorption buffer 1. Add 200 µl adsorption solution to the beads pellet from 2.7. and resuspend. 2. Magnetic separation for 2 min, discard the supernatant. 3. Wash the beads in Adsorption buffer two more times. 4) Adsorption of protein/peptides 1. Add your sample containing approx. 10 µg protein or peptide to the washed M-Beads Magnetic silica beads WAX and add adsorption solution to a total volume of 100 µl. 2. Leave the beads at room temperature for about 5 min. for proper adsorption of the sample. Continuous shaking is of advantage. 3. Magnetic separation until the liquid is totally clear, discard the supernatant 4. Remove the tube from the magnet and add 200 µl adsorption solution. 5. Magnetic separation for two minutes, discard the supernatant. 6. Repeat washing (steps 4 + 5) to a total of three times, discard the supernatant 5) Desorption for MS analysis 1. Add 100 µl washing solution to the bead pellet and resuspend (desalting step). 2. Magnetic separation for 2 min., discard the supernatant. 3. Add 10 µl desorption solution 1 to the beads, resuspend. 4. Magnetic separation for 2 min, remove the liquid for further analysis to a fresh Eppendorf tube. Maldi analysis: Typically 1 µl of the eluate and 1 µl of a saturated solution of a proper Maldi-MS matrix are mixed. 1 µl spotted on Maldi target gives sufficient spectra. 5.2) Desorption under native protein conditions 1. Resuspend the beads in the 20 µl desorption solution 2a (0.02 M bis Tris, ph 6, 0.05 M NaCl) and incubate for 2 min. at room temperature. 2. Separate the beads at the magnetic separator and collect the supernatant. 3. Repeat step 1) and 2) with increasing salt concentrations of the desorption solution 2b - 2e). 5.3) Desalting after 5.2. If a desalting is needed after desorption under native conditions (5.2), e.g. for mass spec analysis, we recommend the M-Beads Magnetic silica beads S-C4, S-C8 or S-C18 beads (cat. numbers: PR-MAG00040, PR-MAG00041, PR-MAG00004).

7 MoBiTec GmbH 2012 Page 7 Order Information, Shipping and Storage Order# Product Quantity PR-MAG M-Beads Magnetic silica beads WAX 2 ml PR-MAG M-Beads Magnetic silica beads WAX 10 ml shipped at RT; store at 2-8 C As a complemt product we also recommend to consider M-Beads Magnetic silica beads WCX in your application: Order# Product Quantity PR-MAG M-Beads Magnetic silica beads WCX 2 ml PR-MAG M-Beads Magnetic silica beads WCX 10 ml shipped at RT; store at 2-8 C For desalting after M-Beads Magnetic silica beads WAX fractionation we recommend: Order# Product Quantity PR-MAG M-Beads Magnetic silica beads S-C4 2 ml PR-MAG M-Beads Magnetic silica beads S-C4 10 ml PR-MAG M-Beads Magnetic silica beads S-C8 2 ml PR-MAG M-Beads Magnetic silica beads S-C8 10 ml PR-MAG M-Beads Magnetic silica beads S-C18 2 ml PR-MAG M-Beads Magnetic silica beads S-C18 10 ml shipped at RT; store at 2-8 C Disclaimer For R&D use only. Not for drug, household or other uses. Avoid contact with the suspension buffer. When disposing the suspension buffer, flush with large amounts of water. Contact and Support MoBiTec GmbH Lotzestrasse 22a D Goettingen Germany Customer Service General inquiries & orders Technical Service Product information phone: +49 (0) phone: +49 (0) fax: +49 (0) fax: +49 (0) order@mobitec.com info@mobitec.com MoBiTec in your area: Find your local distributor at