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Small Molecules: Dye and Biotin 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 important for the accurate determination of dye-to-protein ratios and to help reduce background noise. Post-reaction clean-up of protein samples can be done with Zeba Dye and Biotin Removal Spin Columns and 96-Well Filter Plates. They contain a ready-to-use resin that is designed for small molecule removal, such as non-conjugated fluorescent dyes, biotin, unused reducing agents, and crosslinkers, with exceptional protein recovery. Features include:
- Versatility—helps reduce unreacted fluorescent dyes, biotinylation reagents, crosslinkers, and reducing agents from protein solutions
- High recovery—low-binding resin increases 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
Biotin removal
Biotin removal is important in laboratory tests that use biotin-streptavidin binding technology. Excess biotin can occupy binding sites on streptavidin, which can reduce the efficiency of target protein purification and interfere with the results of the tests. High concentrations of biotin can lead to inaccurate results by preventing proper binding of the target molecules. Therefore, removing excess biotin is crucial to help ensure the accuracy and efficiency of these laboratory assays.
Dye and biotin removal selection guide
Product |  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 | 420 µL |
| Volume capacity | 50–120 μL | 400–700 μL | 1–2 mL | 2–4 mL | 40–100 µL |
| Molecular weight cutoffs (MWCOs) | 7 kDa | 7 kDa | |||
| Automation compatible? | No | Yes | |||
| Options | 5 columns 25 columns 2 x 25 columns | 5 columns 25 columns | 5 columns 25 columns | 5 columns 5 x 5 columns | 2 96-well plates 2 x 2 96-well plates |
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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
Higher small molecule removal and protein recovery performance
Thermo Scientific Zeba Dye and Biotin Removal Columns help 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 more biotin removal from protein samples; while Figure 2 demonstrates how these columns enable a good, 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.
Protein recovery
Thermo Scientific Zeba Dye and Biotin Removal Columns help provide efficient recovery of proteins of greater than 7 kDa.
| Protein | MW (kDa) | % Recovery (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.
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 results 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.
Immunofluorescence image quality
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.
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