Small Molecule, 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 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.

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.

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.

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.