Optimization of magnetic bead-based protein purification
Form |
5 % suspension |
| Binding capacity | 32.4 mg/ml (1.2 pmol/µl of a 27 kDa protein) |
| Matrix | 6% agarose, crosslinked, spherical magnetic beads |
| Bead diameter | 20-40 µm (30 µm average) |
| Specificity | Strep-tag®II and Twin-Strep-tag® |
In the protocol and the following information on optimization of magnetic bead purification, “bead volume” refers to the total volume of beads. 20 µl of the 5 % suspension contain 1 µl of beads.
IBA’s protocol for MagStrep Strep-Tactin®XT beads describe the basic steps for magnetic bead protein purification. Depending on factors such as target protein concentration or size, it may be possible to increase the yield by adjusting parameters of IBA’s base protocol like bead volume or incubation time.
The more beads are used, the more target protein can be bound in a short time.
An increase of the incubation time for several minutes can increase the amount of bound protein and consequently the protein yield.
In comparison to small proteins, large proteins need more space to bind whereby binding sites on the beads are not accessible for other proteins. Thus, purifying large proteins requires more beads to purify an equal number of proteins as for small proteins.
In high concentrated samples, protein and bead can quickly find each other. This facilitates the purification of as much protein as possible.
Effect of protein size on binding efficiency
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 2: 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 3: 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.
Effect of bead volume and incubation time on binding efficiency
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 (1.2 nmol/µl beads or 32.4 µg/µl of a 27 kDa protein). Best yield in relation to the bead volume used has been reached if 5x more beads were added in relation to the maximum amount that can be bound to the beads. For example, if the total amount of protein is 64 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 4 & 5: Protein yield increases when a higher bead volume is used, or if the sample is incubated with a low bead volume for a longer period. 250 µl samples of a 27 kDa Twin-Strep-tag® fusion protein were incubated with different bead volumes on the left, or with the same bead volume for different time periods on the right. Protein content of the elution was compared to the protein content at the start.
Effect of protein concentration in the sample on binding efficiency
As a result, higher target protein concentrations are preferable. Samples with low protein concentrations, like cell culture supernatants or low abundant proteins, should be concentrated before applying them to the magnetic beads. We recommend a protein concentration of at least 1 pmol/µl (or 27 ng/µl of a 27 kDa protein). If possible, measure the protein concentration in your starting sample and adjust the bead volume accordingly.
Figure 6 (left): Higher protein concentrations lead to higher protein yield. 250 µl of different concentrated samples of a 27 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.
However, if concentrating the sample is not possible and only working with low concentrated protein remains, a high volume of beads is not beneficial. Our experiment showed that for low concentrated proteins, a reduction of the bead volume increases the yield.
Figure 7 (right): 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 27 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.
