Subscribe our newsletter here and get 10%* off your next purchase! (terms & conditions*)

Optimization of magnetic bead-based protein purification

MagStrep® Strep-Tactin®XT beads are the ideal tool for quick and simple purification of strep-tagged proteins. They can be used for any type of protein, in small-scale experiments or in high-throughput purifications. Their magnetic core enables separation from the supernatant without centrifugation or the use of a column, making the batch purification workflow very straightforward. Additionally, MagStrep® beads can be used to study protein-protein interactions via pull-down assays. The fast and easy purification conserves even weak protein-protein interactions.
Protocol   Video tutorial 
 
Specifications of the MagStrep® Strep-Tactin®XT beads 

 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® 
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.

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

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. 

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 66 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

An important factor during the purification is the ratio between bead volume and the concentration of target protein in the sample. Our experiments showed that 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 to increase the yield. We recommend a protein concentration of at least 1 pmol/µl (or 30 ng/µl of a 30 kDa protein). If possible, measure the target protein concentration in your sample before purification and adjust the bead volume accordingly.   
However, if concentrating your sample is not an option, using a high volume of beads is not beneficial. For low concentrated proteins, a reduction of the bead volume increases the yield. Bead volume and protein concentration must always 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.

FAQ about magnetic bead purification 

Yes, protein purification with magnetic beads can also be performed at lower temperatures.
Yes, the Strep-tag® system is highly adaptable to different buffer compositions, allowing the purification of a range of protein types. Please check out our list of compatible reagents for Strep-Tactin®XT.
A scale-up of magnetic bead purification is possible. However, for larger volumes a stronger magnet is needed to attract all the beads. For the purification of larger volumes, we recommend Strep-Tactin®XT resins in columns.
If you are eluting by boiling the beads, use biotin for elution instead. If the contaminations persist, try adding reducing agents or raising the ionic strength of the buffers.
No, the affinity of Strep-Tactin®XT to biotin is too low for an efficient purification of biotinylated proteins. We recommend our MagStrep® Strep-Tactin® beads which are suitable for the purification of biotinylated proteins.
No, MagStrep® Strep-Tactin®XT beads are not suitable for cell isolation due to their large diameter. We recommend our Strep-Tactin® magnetic microbeads which have a diameter of 1-3 µm and can be used to isolate cells via Strep-tagged Fab fragments or other tagged proteins with an affinity towards the cells.