Sodium dodecyl sulfate is one of the most used detergents for solubilizing biological materials. Still, excess unbound detergent interferes with many downstream applications like mass spectrometry (MS) and amino acid sequencing, antigen-antibody binding, immunoprecipitation assay, and ELISA. Several SDS removal protocols, such as prolonged dialysis, anion exchange chromatography, spin column, and acetone precipitation, are routinely used. However, these procedures are either laborious or suffer from sample losses and are challenging for low volume samples and high thorough-put automation. We developed a novel, efficient SDS removal system to overcome these limitations.
BcMag™ One-Step SDS Removal Kit uses magnetic beads modified with proprietary chemistry to remove SDS detergent. The resin can quickly and efficiently remove free SDS (sodium dodecyl sulfate) from ultra-low volumes of protein/ peptide or DNA/RNA solutions. The beads enable 96 samples to be processed simultaneously in less than 10 minutes.
The beads allow rapid and efficient removal of free SDS from the sample. The procedure is straightforward (Fig.1). 1. Add the beads directly to the sample. 2. Pipette or vortex to capture the free SDS detergent. 3. Magnetic separation of the beads from the protein, or DNA/RNA solution, while the protein or DNA/RNA remains in the solution. The easy-to-use magnetic beads significantly improve results over the standard drip column and batch methodologies with minimum protein loss (<10%). Since only a small volume of magnetic beads is used, the final protein concentration of the sample is not significantly decreased.
Features and Advantages
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Simple protocol: No liquid transfer, One-tube, One-step, and one-minute protocol
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Easy-to-use
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Reliable and reproducible results with exceptional >90% recovery for protein (>6 kDa, aprotinin) or DNA/RNA (>25mer dsDNA)
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Effective Cleanup: Remove 95% free SDS detergent
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Cost-effective: Eliminates columns, filters, and laborious repeat pipetting
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High throughput: Compatible with many different automated liquid handling systems
PROTOCOL
Materials Required by the User
Item
Magnetic Rack for centrifuge tube
** Based on sample volume, the user can choose one of the following magnetic Racks
Source
• BcMag™ Rack-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-01)
• BcMag™ Rack-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-02)
• BcMag™ Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat. No. MS-03)
• BcMag™ Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-04)
Item
BcMag™ 96-well Plate Magnetic Rack.
Source
• BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-05)
Item
Adjustable Single and Multichannel Pipettes
Item
Centrifuge with Swinging Bucket
Addition items are required if using 96-well PCR plates / tubes
Vortex Mixer
** The user can also use other compatible vortex mixers. However, the Time and speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm
Eppendorf™ MixMate™
Eppendorf, Cat. No. 5353000529
Tube Holder PCR 96
Eppendorf, Cat. No. 022674005
Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL
Eppendorf, Cat. No. 022674048
Smart Mixer, Multi Shaker
BenchTop Lab Systems, Cat. No. 5353000529
1.5/2.0 mL centrifuge tube
96-well PCR Plates or 8-Strip PCR Tubes
PCR plates/tubes
** IMPORTANT! If using other tubes or PCR plates, make sure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.
Items
Magnetic Rack for centrifuge tube
** Based on sample volume, the user can choose one of the following magnetic Racks
Source
●
BcMag™ Rack-2 for holding two individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-01)
●
BcMag™ Rack-6 for holding six individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-02)
●
BcMag™ Rack-24 for holding twenty-four individual 1.5-2.0 ml centrifuge tubes (Bioclone, Cat. No. MS-03)
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BcMag™ Rack-50 for holding one 50 ml centrifuge tube, one 15 ml centrifuge tube, and four individual 1.5 ml centrifuge tubes (Bioclone, Cat. No. MS-04)
BcMag™ 96-well Plate Magnetic Rack
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BcMa™ 96-well Plate Magnetic Rack (side-pull) compatible with 96-well PCR plate and 96-well microplate or other compatible Racks (Bioclone, Cat. No. MS-05)
Adjustable Single and Multichannel Pipettes
Centrifuge with Swinging Bucket
Addition items are required if using 96-well PCR plates/tubes
Vortex Mixer
** The user can also use other compatible vortex mixers. However, the Time and Speed should be optimized, and the mixer should be: Orbit ≥1.5 mm-4 mm, Speed ≥ 2000 rpm
Eppendorf™ MixMate™
Tube Holder PCR 96
Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL
Smart Mixer, Multi Shaker
Eppendorf, Cat. No. 5353000529
Eppendorf, Cat. No. 022674005
Eppendorf, Cat. No. 022674048
BenchTop Lab Systems, Cat. No. 5353000529
Eppendorf™ MixMate™
Tube Holder PCR 96
Tube Holder 1.5/2.0 mL, for 24 × 1.5 mL or 2.0 mL
Smart Mixer, Multi ShakerEppendorf, Cat. No. 5353000529
Eppendorf, Cat. No. 022674005
Eppendorf, Cat. No. 022674048BenchTop Lab Systems, Cat. No. 5353000529
1.5/2.0 mL centrifuge tube
96-well PCR Plates or 8-Strip PCR Tubes
PCR Plates/Tubes
! IMPORTANT ! If using other tubes or PCR plates, ensure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.
Procedure
The following protocol is an example. The beads and sample volume can be rational Scale-up (or down). Do not use buffers containing organic solvents.
1.
Shake the bottle to resuspend the Magnetic beads until it is homogeneous entirely.
! IMPORTANT ! It is essential to mix the beads before dispensing. Do not allow the beads to sit for more than 2 minutes before dispensing. Resuspend the magnetic beads every 2 minutes.2.
Add an appropriate amount of the magnetic beads to the sample containing free detergent.
! IMPORTANT ! Users need to optimize the beads and free detergents ratio based on the binding capacity (48 µg/mg beads**).** Binding capacity assay condition: Mix with 10 µl magnetic beads (100 mg/ml) with 100 µl protein sample (1:400 dilution of Human serum) containing detergents in 0.1M Sodium phosphate, 0.15M NaCl, pH7.5 buffer, and vortex at 2000 rpm for 5 minutes)3.
Mix the sample with beads for 1-2 minutes by slowly pipetting up and down 20-25 times or vortex for 5 minutes at 2000 rpm for PCR plates or .800 rpm for microplates.
! IMPORTANT ! Users should optimize the speed and time if using a vortex mixer.4.
Place the sample plate or tube on the magnetic separation plate for 30 seconds or until the solution is clear.
5.
Transfer the supernatant to a clean plate /tube while the sample plate remains on the magnetic separation plate. The sample is ready for downstream applications.
C. Troubleshooting
Problem
Low Protein Recovery
Probable Cause
Vortexing time is too long.
Suggestion
If using other digital vortex mixers, the vortex condition such as speed and time has to be optimized.
Problem
Low Protein Recovery
Probable Cause
Using too many magnetic beads
Suggestion
Completely resuspend the magnetic beads and reduce the amounts of the beads.
Problem
Failure to remove detergent.
Probable Cause
Used inappropriate tubes or plates
Suggestion
Ensure that the well diameter at the bottom of the conical section of the Tubes or well of the plate is ≥2.5mm.
Problem
Failure to remove detergent.
Probable Cause
Vortex speed is too slow, or vortex time is too short.
Containing too much SDS in the sample
Suggestion
- Increasing either the speed or time
- If using other digital vortex mixers, the vortex condition such as speed and time has to be optimized.
- Repeat the procedure using more beads
Problem
Probable Cause
Suggestion
Low Protein Recovery
Vortexing time is too long.
If using other digital vortex mixers, the vortex condition such as speed and time has to be optimized.
Using too many magnetic beads
Completely resuspend the magnetic beads and reduce the amounts of the beads.
Failure to remove detergent.
Used inappropriate tubes or plates
Ensure that the well diameter at the bottom of the conical section of the Tubes or well of the plate is ≥2.5mm.
- Vortex speed is too slow, or vortex time is too short.
- Containing too much SDS in the sample
- Increasing either the speed or time
- If using other digital vortex mixers, the vortex condition such as speed and time has to be optimized.
- Repeat the procedure using more beads
Learn More
Instruction Manual
MSDS
Sample Preparation Related Products →General Reference
1.
Puchades M, Westman A, Blennow K, Davidsson P. Removal of sodium dodecyl sulfate from protein samples before matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom. 1999;13(5):344-9.
2.
Ilavenil, S., Al-Dhabi, N.A., Srigopalram, S. et al. Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry. Proteome Sci 14, 11 (2016).
3.
Oscar H. Kapp, Serge N. Vinogradov, Removal of sodium dodecyl sulfate from proteins,Analytical Biochemistry,Volume 91, Issue 1,1978.
4.
Doucette, and A. Crowell, “Precipitation of Detergent-Containing Samples for Top-Down and Bottom-Up Proteomics”, in Proteomics Technologies and Applications. London, United Kingdom: IntechOpen, 2019, Pages 230-235
5.
Hou, H., He, H. & Wang, Y. Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27. Sci Rep 10, 3195 (2020).
6.
Sun D, Wang N, Li L. Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis. J Proteome Res. 2012 Feb 3;11(2):818-28.
