Optimization of magnetic bead-based protein purification
Form |
5% suspension |
| Binding capacity | 25.5 mg/ml (0.85 nmol/µl of a 30 kDa protein) |
| Matrix | 6% agarose, crosslinked, spherical magnetic beads |
| Bead diameter | 30 µm average |
| Specificity | Strep-tag®II and Twin-Strep-tag® |
Optimization parameters
IBA’s protocol for MagStrep® Strep-Tactin®XT beads describe the basic steps for magnetic bead protein purification. Depending on your experimental conditions, it's possible to optimize the purification protocol and increase protein yield by adjusting specific parameters. The following tips and data below will help you optimize magnetic bead protein purification by adjusting bead volume, incubation time, and protein concentration.
1. Protein size
2. Incubation time
An increase of the incubation time for several minutes can increase the amount of bound protein and consequently the protein yield.
3. Bead volume and protein concentration
The more beads are used, the more target protein can be bound in a short time.
In high concentrated samples, protein and bead can quickly find each other. This facilitates the purification of as much protein as possible.
1. Required bead volume depends on the protein size
When working with small proteins, more molecules will bind per bead compared to large proteins. Due to their bigger surface area, large proteins require more space to bind. As a result, the binding capacity may noticeably decline for proteins >90 kDa, which can lead to a lower yield.
To ensure a high yield for large proteins, increase the bead volume to provide a sufficient binding surface. The optimal bead volume for your specific protein can be determined by titration.
Figure 1: Protein yield decreases for large proteins. 250 µl samples of Twin-Strep-tag® fusion proteins of different sizes with a starting protein concentration of 1.7 nmol/µl were incubated with 5 µl beads. Protein purification was carried out according to IBA’s standard protocol. After elution, the total protein content was compared to the protein content at the beginning.
Figure 2: Protein yield of large proteins increases when higher bead volumes are used. 250 µl samples of a 140 kDa protein at 1.7 nmol/µl were incubated with different bead volumes for 10 minutes. Afterwards, the purification was performed according to IBA’s standard protocol. Protein content of the elution was compared to the starting protein content.
2. Increased bead volumes reduce the incubation time
To achieve a higher protein yield in a shorter incubation time, add an excess of beads in relation to the amount of protein and binding capacity of the beads (1.1 nmol/µl beads or 33 µg/µl of a 30 kDa protein). Best yield in relation to the bead volume used is reached if 5x more beads are added in relation to the maximum binding capacity. For example, if the total amount of protein is 51 μg, which theoretically could be bound by 2 μl of beads, add 5x 2 μl = 10 μl beads and incubate for 10 minutes to achieve the best yield.
The maximum yield may also be reached when using a lower bead volume, but the incubation time may be noticeably longer.
Figure 3: When a higher bead volume is used, protein yield can be increased at a short incubation time. 250 µl samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with different bead volumes for the same incubation time. Protein content of the elution was compared to the protein content at the start.
Figure 4: At a low bead volume, protein yield can be increased by incubating for a longer time period. 250 µl samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with the same bead volume for different time periods. Protein content of the elution was compared to the protein content at the start.
3. Sample protein concentration and bead volume must be balanced
Figure 5: Higher protein concentrations lead to higher protein yield. 250 µl of different concentrated samples of a 30 kDa Twin-Strep-tag® fusion protein were incubated with 5 µl beads for 10 minutes. IBA’s standard protocol was carried out afterwards and the final protein content of the elution was compared to the protein content at the start.
Figure 6: A high bead volume does not lead to a high yield when working with low protein concentrations. 250 µl sample containing 0.5 or 1 pmol/µl of a 30 kDa Twin-Strep-tag® fusion protein, were incubated with 1.6 or 5 µl beads for 10 minutes. IBA’s standard protocol was carried out afterwards, and the final protein content of the elution was compared to the protein content at the start.
