Detergents contain a hydrophilic head group and a hydrophobic tail. The hydrophobic moiety usually consists of a hydrocarbon chain, while the hydrophilic part has a polar head. Three types of detergents are commonly used in laboratory research, including:

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Non-ionic detergents contain uncharged hydrophilic head groups such as Triton X-100, Triton X-114, NP-40, Tween-20, and Tween-80.

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Ionic detergents are comprised of a hydrophobic chain and anionic or cationic head groups such as sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB).

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Zwitterionic detergents: Those detergents like CHAPS contain negatively and positively charged atomic groups in equal numbers. Therefore, they do not possess a net charge.

Detergents (surfactants) are essential for biomedical research. For successful downstream analysis, it is critical to reduce or entirely remove unbound detergents from the biological sample. However, excess unbound detergent is usually poorly compatible with many downstream applications, including ELISA, protease digestion of proteins, isoelectric focusing, and mass spectrometry (MS). Several commercially available detergent removal methods include prolonged dialysis, anion exchange chromatography, detergent removal spin column, and acetone precipitation. However, these procedures, such as using detergent removal columns, 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 surfactant removal system to overcome these limitations.

BcMag™ One-Step Detergent Removal Kit uses magnetic resin coated with proprietary chemistry to remove detergents. Compared with the detergent removal columns, the resin can quickly and efficiently remove free detergents from the sample with just a single step and enables individual samples or 96 samples to be processed simultaneously in less than 1 minute or 10 minutes with 95% sample recovery. Since the magnetic resin only adsorbs the detergent, the sample recovery rate is exceptional >90%-95%.

Workflow

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 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)

●

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

●

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

Eppendorf, Cat. No. 5353000529

Eppendorf, Cat. No. 022674005

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, ensure that the well diameter at the bottom of the conical section of PCR Tubes or PCR plates must be ≥2.5mm.

Procedure

! IMPORTANT ! 

●

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.

●

The user should optimize the beads and detergent concentration ratio based on the binding capacity listed in Table 1.

Detergent

Binding Capacity**

Protein Recovery (%)

Triton* X-100

17 μg/mg beads

>97

Triton X-114

16.5 μg/mg beads

>96

Tween-20

9 μg/mg beads

>92

NP-40

16 μg/mg beads

>96

Brij-35

16 μg/mg beads

>94

Tween-80

16 μg/mg beads

>93

DDM

15 μg/mg beads

>93

CY-6

16.5 μg/mg beads

>97

CTAB

10 μg/mg beads

>60

** 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)

Procedure

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. 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 need to optimize the beads and free detergents ratio based on the binding capacity listed in Table 1.

• Optimize the Speed and time if using a vortex mixer.

3.

Place the sample plate or tube on the magnetic separation plate for 30 seconds or until the solution is clear.

4.

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 detergent 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 detergent in the sample

• Increasing either the speed or time

• If using other digital vortex mixers, the vortex condition such as Speed and Time must be optimized.

• Repeat the procedure using more beads

Learn More

Instruction Manual

MSDS

General Reference

1.

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).

2.

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.

3.

Yeung YG, Nieves E, Angeletti RH, Stanley ER. Removal of detergents from protein digests for mass spectrometry analysis. Anal Biochem. 2008;382(2):135-137

4.

Antharavally BS, Mallia KA, Rosenblatt MM, Salunkhe AM, Rogers JC, Haney P, Haghdoost N. Efficient removal of detergents from proteins and peptides in a spin column format. Anal Biochem. 2011 Sep 1;416(1):39-44.

5.

W. Holloway. A simple procedure for removal of Triton X-100 from protein samples, Analytical Biochemistry, Volume 53, Issue 1,1973, Pages 304-308.

6.

Stetsenko, A.; Guskov, A. An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization. Crystals 2017