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Small Molecule Removal
Removal of unreacted or residual small molecules in the protein sample preparation workflow is a key success factor for downstream analysis. For example, removal of free dye after a labeling reaction is essential for the accurate determination of dye-to-protein ratios and to help eliminate background noise. The Zeba Dye and Biotin Removal Spin Columns and 96-Well Filter Plates contain a ready-to-use resin that is uniquely designed for rapid removal of non-conjugated fluorescent dyes, biotin, unused reducing agents, and crosslinkers with exceptional protein recovery.
Product highlights
- Versatile—removes unreacted fluorescent dyes, biotinylation reagents, crosslinkers, and reducing agents from protein solutions
- High recovery—low-binding resin maximizes protein recovery
- Easy to use—no cumbersome column preparation or equilibration
- Fast—small-molecule removal and protein recovery in less than 15 mins
- Flexible—available in spin columns and filter spin plates for a range of needs
Applications
- Ideal for post-reaction clean-up of samples
- To effectively remove non-conjugated dye, biotin, reducing agents, and crosslinkers with exceptional protein recovery
Zeba Dye and Biotin Removal Columns and Plates
| Format | 0.5 mL spin columns | 2 mL spin columns | 5 mL spin columns | 10 mL spin columns | Filter plates, 96 well |
|---|---|---|---|---|---|
| Resin bed volume | 0.5 mL | 2 mL | 5 mL | 10 mL | 420uL |
| Volume capacity | 50–120 μL | 400–700 μL | 1–2 mL | 2–4 mL | 40–100 uL |
| Molecular weight cutoffs (MWCOs) | 7 kDa | 7 kDa | 7 kDa | 7 kDa | 7 kDa |
| Automation compatible? | No | No | No | No | Yes |
Product comparison
| Technology / Product | % removal (small molecule) | % recovery (protein) | No instrument required | Versatility | Sample dilution | Ease of use |
|---|---|---|---|---|---|---|
| Zeba Dye and Biotin Removal Columns and Plates |  | | | | | |
| Size exclusion (column chromatography) | |  |  | | | |
| Size exclusion (spin) - BioRad® P30 | | | | | | |
Better small molecule removal and protein recovery performance
Thermo Scientific Zeba Dye and Biotin Removal Columns provide higher biotin removal with excellent protein recovery compared to alternative products.
Figure 1. Zeba Dye and Biotin Removal Columns and similar products from other suppliers (BR = Biorad P-30, GB 1 = G-Bioscience GT-100, GB 6 = G-Bioscience GT-600, GE = PD10) were used to remove 0.27 mM of free NHS LC Biotin from 100 µL samples. Protein recovery of Goat Anti-Mouse (2 mg/mL) labeled with 20X of NHS-LC-Biotin was assessed by Pierce Rapid Gold BCA Assay Kit. Thermo Scientific Biotin Quantitation kit was used to quantitate free biotin removal.
Better immunofluorescence images
Clean up your protein sample by removing unwanted dye, biotin, and crosslinkers. This tool can help you get better immunofluorescence images.
Figure 2. HDAC2 polyclonal antibody was labeled with Alexa Fluor 647 and then purified from unreacted dye using Thermo Scientific Zeba Dye and Biotin Removal Columns. Immunofluorescent analysis of HDAC2 (red) in A549 cells: cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes and blocked with 1% BSA in PBS. Cells were stained with a HDAC2 polyclonal antibody, Alexa Fluor 647 conjugate, with (right panel) and without (left panel) cleanup of unreacted dye at a dilution of 2.5 µg/mL in blocking buffer for 1 hour at room temperature protected from light.
Better performance and time savings
Thermo Scientific Dye and Biotin Removal Columns provide higher dye removal with excellent protein recovery compared to dialysis.
Figure 3 shows how you can achieve exceptional post-reaction protein sample cleanup result in as litle as 15 minutes.
Figure 3. Zeba Dye and Biotin Removal Columns and Thermo Scientific Slide-A-Lyzer G2 Dialysis Cassettes were used to remove free Alexa Fluor 647 Dye from samples of 10 mg/mL Goat Anti-Rabbit IgG labeled with Alexa Fluor 647 (10 molar excess). Protein recovery was assessed by A280 measurements of starting sample and flow through after dye removal. iBright Analysis Software was used to quantitate free dye removal after samples were run on electrophoresis gel and imaged on iBright FL1500 Imaging System.
Excellent protein recovery
Thermo Scientific Zeba Dye and Biotin Removal Columns provide efficient recovery of proteins of greater than 7 kDa.
Excellent dye removal and protein recovery can be achieved by using the Zeba Dye and Biotin Removal Columns.
Table 2. Protein recovery was assessed by applying 1 mg/mL samples of each protein to 5 mL columns. Protein concentrations of processed samples and starting material were quantified using Pierce Rapid Gold BCA Protein Assay Kit.
| % Recovery using 5 mL columns | ||
| Protein | MW (kDa) | 2 mL sample |
|---|---|---|
| Insulin | 5.8 | 29% |
| Cytochrome C | 12 | 91% |
| Chymotrypsin | 25.6 | 97% |
| Protein A | 34 | 97% |
| Transferrin | 80 | 94% |
| Rabbit IgG | 150 | 89% |
Higher reducing agent removal efficiency
Reducing agent removal from protein sample can be done easily and efficiently. Figure 4 compares the efficiency of removing TCEP with Scientific Zeba Dye and Biotin Removal Columns relative to comparable products from other suppliers.
Figure 4. Thermo Scientific Zeba Dye and Biotin Removal Columns and similar products from other suppliers (BR = Biorad P-30, GB 1 = G-Bioscience GT-100, GB 6 = G-Bioscience GT-600, GE = PD10) were used to remove TCEP from of 1 mg/mL goat anti-rabbit IgG containing 25 mM TCEP in PBS. Reducing agent removal was performed by applying 700 µL of sample to 2 mL columns. Quantification of TCEP removal from flow through compared to starting sample was performed using Ellman’s Assay.
Better dye removal performance
Thermo Scientific Zeba Dye and Biotin Removal Columns provide higher dye removal with excellent protein recovery compared to products from other suppliers.
Figure 5 demonstrates how these columns enable a better and simpler way to remove dye from protein samples.
Figure 5. Zeba Dye and Biotin Removal Columns and similar products from other suppliers (BR = Biorad P-30, GB 1 = G-Bioscience GT-100, GB 6 = G-Bioscience GT-600, GE = GE PD10) were used to remove free/unreacted Alexa Fluor 647 Dye from 100 µL samples of 10 mg/mL Goat Anti-Rabbit IgG labeled with Alexa Fluor 647 (10 molar excess). Protein recovery was assessed by A280 measurements of starting sample and flow through after dye removal. iBright Analysis Software was used to quantitate free dye removal after samples were run on electrophoresis gel and imaged on iBright FL1500 Imaging System.
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