MagSi-WAX beads

Catalog No. ABIN1721147

Product Details

Application: Separation (Sep), Sample Preparation (Prep)

Purpose: Peptides and proteins bind to cationic groups on the surface of MagSi-WAX, while impurities are washed away. After elution in Desorption buffer, purified peptides and proteins are ready for downstream use.

Characteristics: The MagSi-WAX (weak anion exchange) magnetic beads are the counter beads to the MagSi WCX beads. Binding, washing and elution are also triggered by the protein/peptide total net charge. The MagSi-WAX beads are ideally suited when more acidic proteins/peptides are expected in the sample. However, our protocols given in the corresponding product sheet cover buffer systems for acidic, neutral and basic conditions.
MagSi-WAX beads are magnetic silica beads with a high magnetic content optimized for protein and peptide separation. Since the MagSi-WCX and MagSi-WAX act as counter-beads, MagnaMedics recommends to test both types of beads, to see which suits best in your individual application.

Components: Magnetic silica beads with fast magnetic separation characteristics, stay long time in suspension.

Material not included: Magnetic separator for bead collection
Mixer/vortex to homogenize samples and re-suspend beads
Buffer as outlined below, pipette tips and suitable tubes

Bead Ligand: Weak Anion Exchange Surface

Bead Matrix: Magnetic Silica particles

Bead Size: Bead size: 1.2 µm

Application Details

Protocol: MagSi-WAX beads are magnetic silica beads coated with a weak anion exchange surface (WAX). 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 lysates
Enrichment of phosphorylated proteins and peptides

MagSi-WAX 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-WAX beads are suitable for use in 96 well microplates on automated liquid handling platforms.

Reagent Preparation:

Buffers and reagents needed
Pre-Load solution: Load MagSi-WAX 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

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 buffers:
0.1 % TFA (pH < 3.0)
sodium citrate buffer, pH 3.5 - 4.5
N-Methylpiperazine, 20 mM, pH 4.5 - 5.0
Piperazine, 20 mM, pH 5.0 - 6.0
bis Tris, 20 mM, pH 5.8 - 6.4
bis Tris propane, 20 mM, pH 6.4 - 7.3
Triethanolamine, 20 mM, pH 7.3 - 7.7

For the corresponding desorption solutions (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.

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. We recommend to use up to 8 mM n-octylglucoside for serum analysis.

Assay Procedure:

Pre-loading with Counter Ions:
1. Vortex MagSi-WAX beads 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 Pre-load solution and re-suspend
7. Magnetic separation 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 resuspend
2. Magnetic separation for 2 min, discard the supernatant
3. Wash the beads in Adsorption Solution two more times

Adsorption of Protein/Peptides:
1. Add your sample containing approx. 10 µg protein or peptide to the washed MagSi-WAX beads and add Adsorption Solution to a total volume 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

Desorption:
A) Desorption for MS analysis:
1. Add 100 µL Washing Solution to the bead pellet and re-suspend (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 (0.02 M Tris, pH 6, 0.05 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 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.

Restrictions: For Research Use only

Handling

Format: Liquid

Concentration: 20 mg/mL

Buffer: Filtered demineralized water

Handling Advice: Store beads in well closed vial and in upright position to prevent drying of the beads.
Do not freeze the product!
Vortex bead suspension well before use.

Storage: 4 °C

Expiry Date: 12 months