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Protocol
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MagSi-WCX beads are magnetic silica beads coated with a weak cation exchange surface (WCX). The beads are intended for:
sample preparation and pre-fractionation prior to mass spectrometry (e.g. MALDI-TOF analysis) and HPLC
protein and peptide separation for multiple downstream applications, e.g. enzymatic assays
detergent removal
Fractionation of clinical samples e.g. serum, plasma, tissues, CSF, urine and cell lysate
MagSi-WCX enables easy handling in both manual and automated workflows. The high magnetic strength of the beads typically results in complete collection in less than 1 minute when magnetic force is applied. Fast and complete separation results in very good reproducibility since no beads will be lost during washing steps. In addition, short incubation times for protein adsorption, desorption and magnetic collection typically significantly decreases the protocol time over conventional column based ion exchange chromatography, e.g. HPLC .
MagSi-WCX beads are suitable for use in 96 well microplates on automated liquid handling platforms.
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Reagent Preparation
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Buffers:
Pre-load solution: Load WCX beads with counter ions: 0.05 M Ammonium Acetate (AmAc) pH 4 plus 1 M NaCl
Adsorption solution: 0.05 M AmAc, pH 4
Washing solution: water (HPLC grade)
Desorption solution 1 for MALDI MS: 1% TFA in water
Desorption solution 2:
a) 0.05 M AmAc, pH 4, 0.2 M NaCl
b) 0.05 M AmAc, pH 4, 0.4 M NaCl
c) 0.05 M AmAc, pH 4, 0.6 M NaCl
d) 0.05 M AmAc. pH 4, 0.8 M NaCl
e) 0.05 M AmAc, pH 4, 1 M NaCl
For buffer systems, the pI of your target molecule should be taken into account. For efficient adsorption and desorption, the pH of the adsorption and desorption buffer should be at least one pH unit below the pI of the molecule to be bound, but should not exceed 4 pH units.
For analysing body fluids like serum, we recommend to test further buffer systems at different pH as well, since typically the pI of the target molecule(s) are unknown.
Optional adsorption and desorption buffers:
50 mM Sodium citrate, pH 5 (with increasing NaCl concentrations as for the AmAc system)
50 mM MES, pH 6
50 mM sodium phosphate pH
50 mM HEPES pH 8
Detergents:
For better handling detergents like 0.01 % Tween 20 or 0.01 % TX-100 might be used. However, please note that detergents might interfere with downstream applications like mass spectrometry. MagnaMedics recommends to use up to 8mM n-octylglucoside for serum analysis.
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Assay Procedure
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Pre-loading with Counter Ions:
1. Vortex MagSi-WCX beads to a homogeneous suspension.
2. Transfer 20 μL beads to an Eppendorf tube.
3. Place the tube to the magnet for 2 minutes.
4. Remove the supernatant.
5. Remove the tube from the magnet.
6. Add 200 μL Pre-load Solution and re suspend.
7. Place the tube to the magnet for 2 minutes, discard the supernatant
8. Repeat step 6 and 7 two times.
Equilibration to Adsorption buffer:
1. Add 200 μL Adsorption Solution to the bead pellet and re-suspend.
2. Magnetic separation for 2 min, discard the supernatant.
3. Wash the beads in Adsorption Solution 2x more.
Adsorption of Protein/Peptides:
1. Add your sample containing approx. 10 μg protein or peptide to the washed MagSi-WCX beads and add Adsorption Solution to a total volume 200 μL.
2. Leave the beads at room temperature for about 5 min. for proper adsorption of the sample. Continuous shaking is of advantage.
3. Place on the magnetic separator 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.
Desorption :
A) 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, re-suspend
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 is mixed (typically, alpha-cyano-4-hydroxy-cinnamic acid is used for peptides < 4000 Da, for proteins > 4000 Da, sinapinic acid is used). Spotting of 1 μL of the mixture on a MALDI target generates reliable spectra.
B) Desorption under native protein conditions:
1. Re-suspend the beads in the 20 μL Desorption Solution 2a (0.05M AmAc, pH 4, 0.2 M NaCl). And incubate for 2 min. at room temperature.
2. Separate the beads at the magnetic separator and transfer the supernatant
3. Repeat step 1) and 2) with increasing salt concentrations of the Desorption Solution 2 b-e)
Desalting after 4B:
If a desalting is needed after desorption under native conditions (4B), e.g. for mass spec analysis, we recommend the MagSi-proteomics C4, C8 or C18 beads
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