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 under 15 mins
- Flexible—available in spin columns and filter spin plates for a range of needs
Applications
- Post-reaction clean-up of protein samples
- Removal of non-conjugated dye, biotin, reducing agents, and crosslinkers
Zeba Dye and Biotin Removal Columns and Plates
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| Zeba Dye and Biotin Removal Spin Columns | Zeba Dye and Biotin Removal Filter Plates | |||
| 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 | |||
| Automation compatible? | No | Yes | |||
| See all Zeba Dye and Biotin Removal Spin Columns and Filter Plates › | |||||
Small molecule removal and protein recovery 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 | | | | | | |
Table 1. Comparison of products used for small molecule removal and protein recovery. Zeba Dye and Biotin Removal Columns and Plates were compared to size exclusion (column chromatography) and size exclusion (spin) from a competitor. This table showcases various properties associated with each technology/product.
Zeba dye and biotin removal product performance
Better small molecule removal and protein recovery performance
Thermo Scientific Zeba Dye and Biotin Removal Columns provide higher biotin removal and higher dye removal with excellent protein recovery compared to alternative products. Additionally, reducing agent removal from protein samples can be performed easily and efficiently. Figure 1 demonstrates how these columns allow for better biotin removal from protein samples; while Figure 2 demonstrates how these columns enable a better and simpler way to remove dye from protein samples. Figure 3 compares the efficiency of removing TCEP with Scientific Zeba Dye and Biotin Removal Columns relative to comparable products from other suppliers.
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.
Figure 2. 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 separated via electrophoresis gel and imaged on the iBright FL1500 Imaging System.
Figure 3. 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 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.
Excellent protein recovery
Thermo Scientific Zeba Dye and Biotin Removal Columns provide efficient recovery of proteins of greater than 7 kDa.
| % 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% |
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.
Better performance and time savings
Thermo Scientific Dye and Biotin Removal Columns provide higher dye removal with excellent protein recovery compared to dialysis. Figure 4 shows exceptional post-reaction protein sample cleanup result in as little as 15 minutes.
Figure 4.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.
Better immunofluorescence images
Zeba dye and biotin removal columns can help clean up protein sample by removing unwanted dye, biotin, and crosslinkers. Figure 5 shows how the removal of unreacted dye can improve the quality of immunofluorescence images.
Figure 5. 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.
Resources
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