{"id":1826,"date":"2018-03-28T09:43:47","date_gmt":"2018-03-28T09:43:47","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=1826"},"modified":"2023-03-02T12:57:20","modified_gmt":"2023-03-02T12:57:20","slug":"streptag","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/testing-procedures\/streptag\/","title":{"rendered":"Streptag"},"content":{"rendered":"

Introduction<\/strong><\/h3>\n

The Streptag II is a short peptide<\/span> (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin, an engineered streptavidin. The Strep-tag II allows affinity chromatography on immobilized Strep-Tactin under physiological conditions, enabling native, active Strep-tagged proteins can be purified in a single step. The binding affinity of the Strep-tag II to Strep-Tactin (Kd = 1 \u03bcM) is nearly 100 times higher than to streptavidin. After a short washing step, gentle elution of purified recombinant protein is performed by addition of low concentrations of biotin or desthiobiotin. Desthiobiotin is a stable, reversibly binding analog of biotin, the natural ligand of streptavidin. Proteins containing the Strep-tag II epitope can be detected with high specificity and sensitivity using Strep-tag Antibodies.<\/p>\n

Construct screening for the best expression template<\/strong><\/h3>\n

Because it contains just 8 amino acids (WSHPQFEK), the Strep-tag has a minimal effect on protein structure and function. However, expression<\/span>-clone screening experiments have shown that addition of a Strep-tag to a recombinant protein construct can increase expression<\/span> and solubility for specific proteins. The tag position can have an effect on expression<\/span>; a tag can be required at the N-terminus to avoid secondary mRNA structures that inhibit initiation of translation. Both examples show that construct screening is a valuable tool for identifying the best expression<\/span> template.<\/p>\n

Purification scale<\/strong><\/h3>\n

The purification scale is dependent on the amount of protein in the preparation. A column size and total binding capacity should be chosen to approximately match the amount of protein to be purified. Very few nontagged proteins will be retained on the resin when nearly all available binding sites<\/span> are occupied by the tagged protein. If too much matrix is used, other proteins may bind nonspecifically to unoccupied sites and elute as contaminants.<\/p>\n

High-yield preparations from E. coli<\/span> cultures can be purified using a batch procedure<\/span>. Eukaryotic expression<\/span> systems typically deliver smaller amounts of protein than prokaryotic systems. This requires that smaller amounts of purification matrix be used for high-efficiency purification. Proteins that are obtained in high yields from baculovirus-infected insect cells can be purified using batch procedures. However, if protein expression<\/span> is low, a purification procedure<\/span> using magnetic beads is the method of choice. Strep-tagged proteins expressed in mammalian cells should be purified using Strep-Tactin Magnetic Beads.<\/p>\n

Matrix \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Protein<\/span> binding capacity<\/p>\n

Strep-Tactin Superflow\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ~1 mg\/ml (~50 nmol @ ~20 kDa)<\/p>\n

Strep-Tactin Magnetic Beads \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u00a0\u2013300 \u03bcg\/ml (10% suspension)<\/p>\n

(~10\u201315 nmol @ ~20 kDa)<\/p>\n

Buffer and reagent compatibility<\/strong><\/h3>\n

Some reagents and buffer components used for protein purification may interfere with the affinity interaction between the protein and purification matrix.<\/p>\n

Reagents compatible with the Strep-tag\u2013Strep-Tactin interaction*<\/p>\n

250 mM imidazole, 5 mM DTT, 1% Triton\u00ae X-100 1 M NaCl, 1% Tween\u00ae20, 10% glycerol<\/span>, 0.3% CHAPS, 1 mM EDTA and 2% Igepal CA-630 (Nonidet P40).<\/p>\n

Protocol: Growth of E. coli Cultures (50 ml)<\/strong><\/h3>\n

Materials<\/strong><\/h3>\n
    \n
  • LB medium<\/li>\n
  • Antibiotics<\/li>\n
  • IPTG (Isopropyl \u03b2-D-1-thiogalactopyranoside)<\/li>\n<\/ul>\n

    Procedure<\/strong>\u00a0<\/strong><\/h3>\n
      \n
    1. Inoculate 10 ml of LB medium containing the appropriate antibiotics in a 50 ml flask. Grow the cultures at 37\u00b0C overnight.<\/li>\n
    2. Inoculate 50 ml of prewarmed media (with appropriate antibiotics) with 2.5 ml of the overnight cultures and grow at 37\u00b0C with vigorous shaking until an OD600 of 0.6 is reached (30\u201360 min).<\/li>\n
    3. Take a 0.5 ml sample immediately before induction. This sample is the noninduced control; pellet cells and resuspend them in 25 \u03bcl 5x SDS-PAGE sample buffer. Freeze and store the sample at \u201320\u00b0C until SDS-PAGE analysis.<\/li>\n
    4. Induce expression<\/span> by adding IPTG to a final concentration of 1 mM.<\/li>\n
    5. Incubate the cultures for an additional 4\u20135 h. Collect a second 0.5 ml sample. This sample is the induced control; pellet cells and resuspend them in 50 \u03bcl 5x SDS-PAGE sample buffer. Freeze and store the sample at \u201320\u00b0C until SDS-PAGE analysis.<\/li>\n
    6. Harvest the cells by centrifugation<\/span> at 4000 x g for 20 min.<\/li>\n
    7. Freeze and store cell pellet overnight at \u201320\u00b0C.<\/li>\n<\/ol>\n

      Protocol: Preparation of Cleared Lysates from E. coli Cell Cultures<\/strong><\/h3>\n

      This lysis protocol uses Strep-Tactin Superflow Lysis Buffer<\/span> and is suitable for subsequent single-step purification using a Strep-Tactin matrix.<\/p>\n

      For ultrahigh-purity preparations we recommend a two-step purification procedure<\/span> using Ni-NTA Superflow followed by further purification using Strep-Tactin Superflow. In such cases, use Ni-NTA Superflow Lysis Buffer<\/span> for cell lysis. The amount of cells required depends on the expression<\/span> level of the Strep-tagged protein. For proteins that are expressed at high levels (10\u201350 mg of tagged protein per liter of cell culture), a 10x concentrated cell lysate (resuspend the pellet from a 50 ml culture in 5 ml lysis buffer<\/span>) can be used.<\/p>\n

      Four ml of a 10x concentrated cell lysate in lysis buffer<\/span> will contain approximately 0.4\u20132 mg of tagged protein.<\/p>\n

      For much lower expression<\/span> levels (1\u20135 mg\/liter), 200 ml of cell culture should be used to obtain a 50x concentrated cell lysate (4 ml cell lysate = 0.2\u20131 mg of tagged protein).<\/p>\n

      Materials<\/strong><\/h3>\n
        \n
      • 50 or 200 ml culture cell pellet<\/li>\n
      • Strep-Tactin Superflow Lysis Buffer<\/span><\/li>\n
      • Lysozyme<\/span><\/li>\n
      • Nuclease<\/li>\n<\/ul>\n

        Procedure<\/strong>\u00a0<\/strong><\/h3>\n
          \n
        1. Thaw the cell pellet for 15 min on ice and resuspend the cells in 5 ml lysis buffer<\/span>.<\/li>\n
        2. Add lysozyme<\/span> to 1 mg\/ml and incubate on ice for 30 min.<\/li>\n
        3. Sonicate on ice. Use six 10 s bursts at 200\u2013300 W with a 10 s cooling period between each burst. Use a sonicator equipped with a microtip.<\/li>\n
        4. If the lysate is very viscous, add Nuclease (3 U per ml culture volume) and incubate on ice for 10\u201315 min. Alternatively, draw the lysate through a blunt-ended, narrow-gauge syringe needle several times.<\/li>\n
        5. Centrifuge lysate at 10,000 x g for 20\u201330 min at 4\u00b0C to pellet the cellular debris. Save supernatant. A certain proportion of the cellular protein may remain insoluble and will be located in the pellet.<\/li>\n
        6. Add 5 \u03bcl 2x SDS-PAGE sample buffer to a 5 \u03bcl aliquot of the supernatant and store at \u201320\u00b0C for SDS-PAGE analysis.<\/li>\n
        7. Proceed using one of the purification protocols.<\/li>\n<\/ol>\n

          Protocol: Preparation of Cleared Lysates from Baculovirus-Infected Insect Cells<\/strong><\/h3>\n

          Since they provide post-translational modifications, insect cells are often the eukaryotic expression<\/span> system of choice. Although expression<\/span> rates are normally significantly higher in insect cells than in mammalian cells, expressed-protein levels are typically lower than those obtained in bacterial<\/span> systems. The estimated total protein content in insect cells is approximately 20 mg per 107<\/sup> cells. With recombinant protein expression<\/span> levels ranging between 0.05% and 50%, protein yields are typically 10 \u03bcg \u2013 10 mg per 107<\/sup> cells. If the expected yield of the preparation is less than 50 \u03bcg, it is highly recommended that proteins be purified using the protocols for Strep-Tactin Magnetic Beads. Larger amounts of protein can be purified using a batch procedure<\/span>. Lysis Buffer<\/span> supplemented with 1% Igepal\u00ae CA-630\u00a0 (Nonidet P40) is used for cell lysis. This lysis protocol uses Strep-Tactin Superflow Lysis Buffer<\/span> and is suitable for subsequent single-step purification using a Strep-Tactin matrix.<\/p>\n

          For ultrahigh-purity preparations we recommend a two-step purification procedure<\/span> using Ni-NTA Superflow followed by further purification using Strep-Tactin Superflow. In such cases, use Ni-NTA Superflow Lysis Buffer<\/span> for cell lysis.<\/p>\n

          Materials<\/strong><\/h3>\n
            \n
          • Cell pellet<\/li>\n
          • PBS<\/li>\n
          • Strep-Tactin Superflow Lysis Buffer<\/span> (Buffer NP) supplemented with 1% Igepal\u00ae CA-630<\/li>\n
          • Nuclease<\/li>\n<\/ul>\n

            \u00a0<\/strong>Procedure<\/strong>\u00a0<\/strong><\/h3>\n
              \n
            1. Wash the transfected cells with phosphate<\/span> buffered saline (PBS) and collect them by centrifugation<\/span> for 5 min at 1000 x g.<\/li>\n
            2. Lyse the cells in lysis buffer<\/span> supplemented with 1% Igepal CA-630 by incubating for 10 min on ice. Use 4 ml lysis buffer<\/span> per 1\u20132 x 107<\/sup> cells.<\/li>\n
            3. If the lysate is very viscous, add Nuclease (3 U per ml culture volume) and incubate on ice for 10\u201315 min. Alternatively, draw the lysate through a blunt-ended, narrow-gauge syringe needle several times.<\/li>\n
            4. Centrifuge the lysate at 10,000 x g for 10 min at 4\u00b0C to pellet cellular debris and DNA. Save the cleared lysate (supernatant). The supernatant should contain the tagged protein.<\/li>\n
            5. Proceed using one of the purification protocols.<\/li>\n<\/ol>\n

              Protocol: Batch Purification of Strep-tagged Proteins Using Strep-Tactin Superflow<\/strong><\/h3>\n

              \u00a0<\/strong>The composition of the lysis and elution buffers can be modified to suit the particular application, e.g., by adding 0.1% Tween, 5 mM DTT, or 1 mM PMSF, or increasing NaCl or glycerol<\/span> concentrations.<\/p>\n

              Materials<\/strong><\/h3>\n
                \n
              • Cleared lysate<\/li>\n
              • Strep-Tactin Superflow Lysis Buffer<\/span> (Buffer NP)<\/li>\n
              • Empty columns<\/li>\n
              • Strep-Tactin Superflow Elution Buffer (Buffer NPD)<\/li>\n
              • Strep-Tactin Superflow<\/li>\n<\/ul>\n

                Procedure<\/strong><\/h3>\n
                  \n
                1. Pipet cleared cell lysate into a 15 ml tube and add 2 ml Strep-Tactin Superflow Resin suspension. Mix gently by shaking (200 rpm on a rotary shaker) at 4\u00b0C for 60 min.<\/li>\n
                2. Load the Strep-Tactin Superflow resin into a column with the bottom outlet capped.<\/li>\n
                3. Remove bottom cap and collect the flow-through. Save flow-through for SDS-PAGE analysis.<\/li>\n
                4. Wash column two times with 4 ml Strep-Tactin Superflow Lysis Buffer<\/span>. Collect wash fractions for SDS-PAGE analysis.<\/li>\n
                5. Elute the protein six times with 0.5 ml Strep-Tactin Superflow Elution Buffer. Collect eluates in six tubes and analyze by SDS-PAGE.<\/li>\n<\/ol>\n

                  Protocol: Micro-Scale Purification of Strep-tagged Proteins Using Strep-Tactin Magnetic Beads<\/strong><\/h3>\n

                  The composition of the lysis and elution buffers can be modified to suit the particular application, for example by adding 0.1% Tween, 5 mM DTT, or 1 mM PMSF, or increasing NaCl or glycerol<\/span> concentrations.<\/p>\n

                  The amount of tagged protein recovered using Strep-Tactin Magnetic Beads is proportional to the protein concentration in the sample. Therefore, if samples contain low concentrations of protein, we recommended removing the storage buffer from the Strep-Tactin Magnetic Beads before adding the sample to the beads.<\/p>\n

                  Materials<\/strong><\/h3>\n
                    \n
                  • Cleared lysate<\/li>\n
                  • Strep-Tactin Magnetic Beads<\/li>\n
                  • Strep-Tactin Beads Lysis Buffer<\/span><\/li>\n
                  • Strep-Tactin Beads Elution Buffer<\/li>\n
                  • Optional: Benzonase\u00ae Nuclease<\/li>\n<\/ul>\n

                    Procedure<\/strong><\/h3>\n
                      \n
                    1. Resuspend Strep-Tactin Magnetic Beads by vortexing for 2 s and then immediately add 200 \u03bcl of 10% Strep-Tactin Magnetic Bead suspension to a cleared lysate or the pooled fractions eluted from Ni-NTA Magnetic Beads. Care is necessary to ensure that constant amounts of beads are pipetted. The beads will settle if the suspension is not agitated regularly. 200 \u03bcl of Strep-Tactin Magnetic Beads suspension has a binding capacity of 40\u201360 \u03bcg protein<\/span>. If significantly different amounts of tagged protein are present in your lysate, the volume of magnetic-bead suspension should be varied accordingly. However, use of volumes less than 10 \u03bcl are not recommended due to the associated handling problems \u2014 smaller volumes are difficult to pipet and may lead to uneven distribution of beads and reduced reproducibility. Generally, we recommend using cleared lysates for binding to Strep-Tactin Magnetic Beads. However, it may be possible to obtain good results by using crude lysates without clearing them. In this case, add Nuclease (3 U per ml culture volume), and incubate on ice for 10\u201315 min.<\/li>\n
                    2. Mix the suspension gently on an end-over-end shaker for 30\u201360 min at 4\u00b0C.<\/li>\n
                    3. Place the tube on a magnetic separator for 1 min and remove supernatant with a pipet. Tubes may be briefly centrifuged, before placing on the magnetic separator, to collect droplets of suspension from the tube caps.<\/li>\n
                    4. Remove the tube from the magnet, add 500 \u03bcl Strep-Tactin Beads Lysis Buffer<\/span>, gently vortex the suspension, place the tube on a magnetic separator for 1 min, and remove buffer.<\/li>\n
                    5. Repeat step 4. Remaining buffer should be removed completely.<\/li>\n
                    6. Add 50 \u03bcl Strep-Tactin Beads Elution Buffer, gently vortex the suspension, incubate the tube for 5 min, place the tube on a magnetic separator for 1 min, and collect the eluate in a clean tube. Tubes may be centrifuged before placing on the magnetic separator, to collect droplets of suspension from the tube caps.<\/li>\n
                    7. Repeat step 6 three times to give four eluate fractions. Because the biotin used for elution binds Strep-Tactin with extremely high affinity, it is not possible to regenerate Strep-Tactin Magnetic Beads<\/li>\n<\/ol>\n

                      Protocol: Immunodetection using the Strep-tag Antibody (Chemiluminescent Method)<\/strong><\/h3>\n

                      Materials<\/strong><\/h4>\n
                        \n
                      • Western blot or dot blot<\/li>\n
                      • TBS (10 mM Tris\u00b7Cl; 150 mM NaCl, pH<\/span> 7.5)<\/li>\n
                      • TBS-Tween\/Triton (20 mM Tris\u00b7Cl; 500 mM NaCl; 0.05% (v\/v) Tween; 0.2% (v\/v) Triton, pH<\/span> 7.5)<\/li>\n
                      • Blocking buffer (3% BSA or 1% casein<\/span> in TBS)<\/li>\n
                      • Strep-tag Antibody stock solution: Dissolve the lyophilized Strep-tag Antibody in 500 \u03bcl water. The reconstituted solution contains 200 \u03bcg\/ml Strep-tag Antibody in PBS with PEG, sucrose, and sodium azide (0.08% [w\/v]).<\/li>\n
                      • Anti-mouse secondary antibody<\/span> conjugate<\/li>\n
                      • Secondary antibody<\/span> dilution buffer (10% milk powder or 1% casein<\/span> in TBS)<\/li>\n<\/ul>\n

                        For chemiluminescent detection, BSA does not sufficiently block nonspecific binding of the secondary antibody<\/span> to the membrane, and milk powder should be used to dilute the secondary antibody<\/span>. Alternatively, if alkali-soluble casein<\/span> is available it can be used as a blocking reagent throughout the chemiluminescent detection protocol.<\/p>\n

                        Reagents used in chemiluminescent detection of Strep-tagged proteins<\/p>\n

                        Step \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 This Method \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 (Alternative method)<\/strong><\/p>\n

                        Blocking \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 3% BSA in TBS \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1% casein<\/span> in TBS<\/p>\n

                        Strep-tag Antibody binding\u00a0 3% BSA in TBS \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 1% casein<\/span> in TBS<\/p>\n

                        Secondary antibody<\/span> binding 10% milk powder in TBS\u00a0\u00a0\u00a0 1% casein<\/span> in TBS<\/p>\n

                        Procedure<\/strong><\/h3>\n
                          \n
                        1. Wash membrane twice for 10 min each time with TBS buffer at room temperature (15\u201325\u00b0C).<\/li>\n
                        2. Incubate for 1 h in blocking buffer at room temperature. 3% BSA (w\/v) in TBS buffer*, is used for blocking until incubation.<\/li>\n
                        3. Wash membrane twice for 10 min each time in TBS-Tween\/Triton buffer at room temperature.<\/li>\n
                        4. Wash membrane for 10 min with TBS buffer at room temperature.<\/li>\n
                        5. Incubate with Strep-tag Antibody (1\/1000\u20131\/2000 dilution of antibody stock solution in blocking buffer) at room temperature for 1 h. Membrane can be sealed in plastic bags. 3% BSA (w\/v) in TBS buffer* is used for this blocking step when using chemiluminescent detection.<\/li>\n
                        6. Wash twice for 10 min each time in TBS-Tween\/Triton buffer at room temperature.<\/li>\n
                        7. Wash for 10 min in TBS buffer at room temperature.<\/li>\n
                        8. Incubate with secondary antibody<\/span> solution for 1 h at room temperature. Either alkaline phosphatase (AP) or horseradish peroxidase (HRP) conjugated anti-mouse IgG may be used. Dilute according to the manufacturer\u2019s recommendations. Use the lowest recommended concentration to avoid false signals. 10% nonfat dried milk in TBS* is used for incubation with secondary antibody<\/span> when using chemiluminescent detection. Milk powder is needed to reduce background because BSA does not block sufficiently for the very sensitive chemiluminescent detection method.<\/li>\n
                        9. Wash 4 times for 10 min each time in TBS-Tween\/Triton buffer at room temperature.<\/li>\n
                        10. Perform chemiluminescent detection reaction and expose to X-ray film according to the manufacturer\u2019s recommendations.<\/li>\n<\/ol>\n

                           <\/p>\n","protected":false},"excerpt":{"rendered":"

                          Introduction The Streptag II is a short peptide (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin, an engineered streptavidin. The Strep-tag II allows affinity chromatography on immobilized Strep-Tactin under physiological conditions, enabling native, active Strep-tagged proteins can be purified in a single step. The binding affinity of the Strep-tag II to Strep-Tactin […]<\/p>\n","protected":false},"author":4,"featured_media":0,"parent":401,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-1826","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1826","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=1826"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1826\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/401"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=1826"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}