Pierce Dye and Biotin Removal Spin Columns and 96-Well Filter Plates

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 Pierce 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

Choose the right biotin and dye removal product for your experiment

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) 7KDa 7KDa 7KDa 7KDa 7KDa
Automation compatible? No No No No Yes

Table 1. Compare available product options for small molecule removal and protein recovery

Technology / Product % removal (small molecule) % recovery (protein) Time saving No instrument required Versatility Sample dilution Ease of use
Pierce dye and biotin Removal columns and plates
Size exclusion (column chromatography)
Size exclusion (spin) - BioRad® P30
Ion exchange (Pierce Dye Removal Columns)
Dialysis (Slide-a-Lyzer Cassettes)

Better small molecule removal and protein recovery performance

Thermo Scientific Pierce Dye and Biotin Removal Columns provide higher biotin removal with excellent protein recovery compared to alternative products.

Better immunofluorescence images

Clean up your protein sample by removing unwanted dye, biotin, and crosslinkers. This tool will help you get better immunofluorescence images.

HDAC2 polyclonal antibody labeled with Alexa Fluor 647
Figure 2. HDAC2 polyclonal antibody (Cat. No. PA1-861) was labeled with Alexa Fluor 647 (Cat. No. A20006) and then purified from unreacted dye using Thermo Scientific Pierce Dye and Biotin Removal Columns (Cat. No. A44296). 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 superior post-reaction protein sample cleanup result in 15 minutes.

Excellent protein recovery

Thermo Scientific Pierce Dye and Biotin Removal Columns (Cat. No. A44298) provide efficient recovery of proteins of greater than 7 kDa.

Excellent dye removal and protein recovery can be achieved by using the Pierce Dye and Biotin Removal Columns.

Table 2. Protein recovery was assessed by applying 1 mg/mL samples of each protein to 5 mL columns (Cat. No. A44300). Protein concentrations of processed samples and starting material were quantified using Pierce Rapid Gold BCA Protein Assay Kit (Cat. No. A53226).

  % 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 Pierce Dye and Biotin Removal Columns relative to comparable products from other suppliers.

Better dye removal performance

Thermo Scientific Pierce 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.

Bar graph showing greater dye removal and comparable protein recovery for the Pierce Dye and Biotin Removal Columns relative to other suppliers
Figure 5. Pierce Dye and Biotin Removal Columns (Cat. No. A44296) 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 (Cat. No. A20006) 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 (Cat. No. A44241).