{"id":1816,"date":"2018-03-27T20:09:38","date_gmt":"2018-03-27T20:09:38","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=1816"},"modified":"2023-03-02T13:11:44","modified_gmt":"2023-03-02T13:11:44","slug":"the-streptamer-principle","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/testing-procedures\/the-streptamer-principle\/","title":{"rendered":"The Streptamer Principle"},"content":{"rendered":"

Strep<\/em>-tags are short peptides with high binding selectivity for Strep<\/em>-Tactin, an engineered streptavidin. The binding affinity<\/span> of e.g. Strep<\/em>-tag II to Strep<\/em>-Tactin (Kd = 1 \u00b5M) is nearly 100 times higher than to streptavidin. Strep<\/em>-tags may be fused to recombinant proteins<\/span> which allows efficient one-step purification of such fusion proteins<\/span> on immobilized Strep<\/em>– Tactin under physiological conditions, thus preserving their bioactivity.<\/p>\n

As the Strep<\/em>-tag binds to the biotin binding pocket of Strep<\/em>-Tactin, purified proteins<\/span> may be mildly eluted from the column by the addition of minute amounts of biotin.<\/p>\n

A special application of the Strep<\/em>-tag:Strep<\/em>-Tactin technology is the oligomerization of MHC<\/span> I-Strep<\/em>-tag fusion proteins<\/span> on Strep<\/em>-Tactin. These complexes may be used for the efficient antigen<\/span> specific staining of T-cells<\/span> by using modified Strep<\/em>-Tactin being labeled by a fluorescent probe or a magnetic particle. After separation of the stained T-cells<\/span> from non-stained cells by florescence activated cell sorting (FACS) or by a magnetic field, respectively, the staining may be efficiently removed by the addition of biotin.<\/p>\n

This removal of the Strep<\/em>-Tactin backbone leaves monomeric MHC<\/span> I-Strep<\/em>-tag fusion proteins<\/span> on the surface of the T-cell. As the monovalent MHC<\/span> I:T-cell receptor<\/span> interaction is weak, MHC<\/span> I-Strep<\/em>-tag fusion proteins<\/span> spontaneously dissociate from the T-cell receptor<\/span> and may be removed from the T-cells<\/span> simply by washing. Keeping stained cells at 4 \u00b0C together with rapid and complete dissociation of Strep<\/em>tamers from the T-cells<\/span> after purification assures the isolation of fully functional, non-induced T-cells<\/span>.<\/p>\n

Experimental procedure<\/strong><\/h3>\n

Routinely approximately 5×106<\/sup> cells are stained using 0.75 \u00b5g Strep<\/em>-Tactin-PE (5 \u00b5l) and1 \u00b5g MHC<\/span> I (4 \u00b5l) in a final volume of 50 \u00b5l. All steps, the staining of the cells as well as the following dissociation of Strep<\/em>tamers, have to be performed at 4\u00b0C. Please make sure that all your reagents and the cells have reached the temperature before starting the protocol.<\/p>\n

Titration<\/strong><\/h3>\n

If the staining protocol is not suitable for your application, a titration of the MHC<\/span> should be performed.<\/p>\n

    \n
  • Test 0.75 mg Strep<\/em>-Tactin-PE with 1 mg, 2 mg and 5 mg MHC<\/span> I, respectively.<\/li>\n
  • The assay can be conducted in a 96-well round bottom microplate.<\/li>\n
  • All incubations are carried out in the dark to protect PE from light.<\/li>\n<\/ul>\n

    Protocol for the staining of T-cells with Strep<\/em>tamers<\/strong><\/h3>\n

    All steps have to be performed at 4\u00b0C<\/p>\n

      \n
    1. Incubate 0.75 \u00b5g (5 \u00b5l) Strep<\/em>-Tactin-PE and 1 \u00b5g (4 \u00b5l) MHC<\/span> in a final volume<\/li>\n<\/ol>\n

      of 50 \u00b5l Buffer IS for 45 minutes.<\/p>\n

        \n
      1. Add the pre-incubated Strep<\/em>-Tactin-PE\/MHC<\/span> I preparation to the cell pellet.<\/li>\n
      2. Incubate for 45 minutes.<\/li>\n
      3. Wash cells twice with 200 \u00b5l Buffer IS.<\/li>\n
      4. Cells are ready for FACS-analysis or FACS-sorting.<\/li>\n<\/ol>\n

        Strep<\/em>tamer Magnetic T-cell Labeling and Isolation via MACS<\/p>\n

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

        In a first step, T-cells<\/span> are labeled with a magnetic Strep<\/em>tamer complex according to their antigen<\/span> specificity, then stained T-cells<\/span> are separated from other cells by a magnetic field and such purified T-cells<\/span> are eluted and released from the Streptamer complex by the addition of biotin (vitamin H) to yield a functional, non-induced antigen<\/span> specific T-cell preparation<\/p>\n

        Recommendations for isolating T-cells<\/span> using Strep<\/em>-Tactin magnetic beads and recombinant MHC<\/span> I proteins<\/span> fused to Strep<\/em>tag<\/p>\n

        Experimental procedure<\/strong><\/h3>\n

        The procedure is optimized to isolate antigen<\/span>-specific T-cells<\/span> from 2×107<\/sup> peripheral blood mononuclear cells (PBMC). Some cells like monocytes or natural killer cells may also be co-purified due to their ability to bind MHC<\/span> and can be depleted before the actual T-cell isolation.<\/p>\n

        For human blood:<\/p>\n

          \n
        • Anticoagulant<\/span> treatment<\/li>\n
        • Ficoll gradient<\/li>\n
        • T-cell isolation<\/li>\n<\/ul>\n

          For mouse blood:<\/p>\n

            \n
          • Anticoagulant<\/span> treatment<\/li>\n
          • Ficoll gradient<\/li>\n
          • CD8<\/span>+ enrichment<\/li>\n
          • Optional: NK-cell<\/span> depletion<\/li>\n
          • Antigen<\/span>-specific T-cell isolation<\/li>\n<\/ul>\n

            All the steps from isolation of cells as well as the following dissociation of Strep<\/em>tamers . have to be performed at 4\u00b0C. Please make sure that all your reagents and the cells have reached the temperature before starting the protocol.<\/p>\n

            Reagents<\/strong><\/h3>\n
              \n
            • Strep<\/em>tamer Magnetic Beads:250 \u00b5l (suff. for 1X108<\/sup> cells)<\/li>\n
            • Strep<\/em>tamer Magnetic Beads: 1.25 ml (suff. for 5X108<\/sup> cells)<\/li>\n
            • MHC<\/span> I-Strep<\/em>-tag: 200 \u00b5l (suff. for pur. of 5X108 <\/sup>human or 2.5X108<\/sup>mouse cells)<\/li>\n
            • Solution set for magnetic beads includes: for 5 preps (2X107<\/sup> cells each)<\/li>\n
            • Buffer IS<\/li>\n
            • Biotin stock solution<\/li>\n
            • Nylon filter mesh (100 \u00b5m)<\/li>\n
            • Blood or T-cell sample<\/li>\n
            • Miltenyi columns and separators<\/li>\n
            • Centrifuge<\/li>\n
            • Test tubes<\/li>\n<\/ul>\n

              For optional staining and FACS analysis <\/strong><\/h3>\n
                \n
              • EMA<\/li>\n
              • Strep<\/em>-Tactin PE and recombinant MHC<\/span> I proteins<\/span> fused Strep<\/em>-tag<\/li>\n
              • CD8<\/span>-PE or CD8<\/span>-FITC antibody<\/li>\n
              • CD3-FITC or CD3-APC<\/span> antibody<\/li>\n
              • FACScan<\/li>\n<\/ul>\n

                For optional Ficoll gradient centrifugation<\/span><\/p>\n

                  \n
                • Ficoll<\/li>\n
                • PBS or balanced salt solution<\/li>\n
                • Pasteur pipettes<\/li>\n
                • Syringe with needle<\/li>\n
                • Silicone solution<\/li>\n
                • Distilled<\/span> water<\/li>\n<\/ul>\n

                  For optional CD8<\/span>+ enrichment<\/p>\n

                    \n
                  • CD8<\/span>+ T-cell Isolation Kit<\/li>\n<\/ul>\n

                    Experimental procedure<\/strong><\/h3>\n

                    The procedure is optimized to isolate antigen<\/span>-specific T-cells<\/span> from 2×107<\/sup> peripheral blood mononuclear cells (PBMC). The chapters describe the isolation of these cells and depletion of non-T-cell populations, respectively.<\/p>\n

                    Anticoagulant treatment<\/strong><\/h3>\n
                      \n
                    1. EDTA is added to a final concentration of 20 mM<\/li>\n
                    2. Same volume of PBS or balanced salt solution is added to the EDTA-blood Other anticoagulants have been used like heparin, citrate, acid citrate dextrose, citrate phosphate<\/span> dextrose.<\/li>\n<\/ol>\n

                      Ficoll gradient centrifugation<\/span><\/p>\n

                      Procedure for isolation of lymphocytes<\/span> from blood samples.<\/p>\n

                        \n
                      1. The required volume of Ficoll (3 ml for 4 ml diluted anticoagulated blood sample) is aseptically withdrawn using a syringe.<\/li>\n
                      2. Ficoll-Paque Plus (3 ml) is added to a centrifuge tube<\/li>\n
                      3. Carefully layer diluted blood sample (4 ml) on Ficoll-Paque Plus. When layering the sample do not mix Ficoll and diluted blood sample.<\/li>\n
                      4. Centrifuge at 400x g for 30-40 minutes at 18-20\u00b0C<\/li>\n
                      5. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface. Care should be taken not to disturb the lymphocyte layer. The upper layer of plasma<\/span>, which is essentially free of cells, may be saved for later use.<\/li>\n
                      6. Using a clean pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube. It is critical to remove all of the interface but a minimum amount of Ficoll and supernatant. Removing excess Ficoll causes granulocyte contamination, removing excess supernatant results in platelet<\/span> and plasma<\/span> protein contamination.<\/li>\n
                      7. Add at least 3 volumes of balanced salt solution to the lymphocytes<\/span> in the test tube.<\/li>\n
                      8. Suspend the lymphocytes<\/span> by gently drawing them in and out of a the Pasteur pipette<\/li>\n
                      9. Centrifuge at 10-100 x g and 18-20\u00b0C for 10 minutes.<\/li>\n
                      10. Remove the supernatant<\/li>\n
                      11. The lymphocytes<\/span> should now be suspended in an appropriate medium and can be frozen<\/li>\n<\/ol>\n

                        Optional: CD8+ enrichment<\/strong><\/h3>\n

                        CD8<\/span>+ enrichment is recommended for the isolation of mouse antigen<\/span> specific T-cells<\/span> only.<\/p>\n

                        Magnetic labeling<\/p>\n

                          \n
                        1. Determine cell number<\/li>\n
                        2. Centrifuge cell suspension at 300 x g for 10 minutes. Pipette off supernatant completely<\/li>\n
                        3. Resuspend cell pellet in 40 \u00b5l of buffer per 107<\/sup> total cells.<\/li>\n
                        4. Add 10 \u00b5l of biotin antibody cocktail per 107<\/sup> total cells.<\/li>\n
                        5. Mix well and incubate for 10 minutes at 4-8\u00b0C.<\/li>\n
                        6. Add 30 \u00b5l of buffer per 107<\/sup> total cells.<\/li>\n
                        7. Add 20 \u00b5l of anti biotin MicroBeads per 107<\/sup> total cells.<\/li>\n
                        8. Mix well and incubate for an additional 15 minutes at 4-8\u00b0C.<\/li>\n
                        9. Wash cells with buffer adding 10-20 x labeling volume and centrifuge at 300 x g for 10 minutes. Pipette off supernatant completely.<\/li>\n
                        10. Resuspend cells in 500 \u00b5l buffer. Up to 108<\/sup> total cells can be resuspended in 500 \u00b5l, larger numbers require an accordingly larger volume of buffer.<\/li>\n
                        11. Proceed to magnetic separation.<\/li>\n<\/ol>\n

                          Magnetic separation with MS or LS columns<\/strong><\/h3>\n
                            \n
                          1. Place column in the magnetic field of a suitable MACS separator.<\/li>\n
                          2. Prepare column by rinsing with appropriate amount of buffer: MS: 500 \u00b5l LS: 3 ml<\/li>\n
                          3. Apply cell suspension onto the column. Collect flow-through. Allow cells to pass through and collect effluent as fraction with unlabeled cells, representing the enriched CD8<\/span>+ T-cell fraction.<\/li>\n
                          4. Wash column with appropriate amount of buffer. To wash the column, buffer is added three times when column reservoir is empty: MS: 500 \u00b5l LS: 3 ml. Collect entire effluent and pool with flow-through (step 3). This fraction represents the CD8<\/span>+ T-cells<\/span>.<\/li>\n
                          5. Optional: Elute retained cells outside of the magnetic field. This fraction represents the magnetically labeled non-CD8<\/span>+ T-cells<\/span>.<\/li>\n<\/ol>\n

                            Magnetic separation with the autoMACS separator<\/strong><\/h3>\n
                              \n
                            1. Prepare and prime the autoMACS<\/li>\n
                            2. Place tube containing the magnetically labeled cells in the autoMACS separator and choose program “Deplete”<\/li>\n
                            3. Collect negative fraction. This fraction represents the enriched CD8<\/span>+ T-cells<\/span>.<\/li>\n
                            4. Optional: Collect positive fraction. This fraction represents the magnetically labeled non-CD8<\/span>+ T-cells<\/span>.<\/li>\n<\/ol>\n

                              Isolation of antigen specific T-cells with Strep<\/em>tamer magnetic beads<\/strong><\/h3>\n

                              Protocol for human cells<\/strong><\/h4>\n

                              When working with anti-coagulated peripheral blood or buffy coat, PBMC should be<\/p>\n

                              isolated by density gradient centrifugation<\/span> first. This Protocol is adapted for 2 x 107<\/sup> cells. Higher cell numbers require larger amounts of beads and MHC<\/span>.<\/p>\n

                                \n
                              1. Thaw frozen cells in normal growth medium. Make sure concentration of DMSO is below 1%. Cells grown in medium containing less than 10% FCS should be thawed in medium containing 10% FCS instead of their normal growth medium<\/li>\n
                              2. Wash cells in buffer IS and resuspend in 10 ml (use 300 g for each centrifugation<\/span>).<\/li>\n
                              3. Pass cells through enclosed 100 \u00b5m nylon mesh. This is necessary to remove cell clumps which may clog the columns.<\/li>\n
                              4. Determine cell number, take sample (before separation) and place cells on ice. 5. Incubate 50 \u00b5l magnetic beads, 8 \u00b5l MHC<\/span>, and 90 \u00b5l buffer IS at least 45 minutes at 4\u00b0C (or over night).<\/li>\n
                              5. Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml buffer IS.<\/li>\n
                              6. Add 1 ml buffer IS to MHC<\/span>\/magnetic beads solution and load on MS column. To wash away unbound MHC<\/span>, magnetic beads are bound and washed on a MS column.<\/li>\n
                              7. Wash with 2 ml buffer IS.<\/li>\n
                              8. Add 250 \u00b5l buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column.<\/li>\n
                              9. Centrifuge cell suspension (300 g) and resuspend in 250 \u00b5l magnetic beads\/MHC<\/span> solution. Incubate 45 minutes on ice.<\/li>\n
                              10. Add 1.5 ml buffer IS, centrifuge cell and beads mixture and wash carefully once with 2 ml buffer IS. This is necessary to eliminate unbound magnetic beads which may trap cells on the column un-specifically.<\/li>\n
                              11. Resuspend in 2 ml buffer IS. Proceed to magnetic separation.<\/li>\n<\/ol>\n

                                Isolation of antigen specific T-cells with Strep<\/em>tamer magnetic beads<\/strong><\/h3>\n

                                Protocol for mouse cells<\/strong><\/h4>\n

                                When working with cells from spleen<\/span> or lymph<\/span> node cells, be careful to resuspend cells completely. Other organ preparations may require protease digestion and\/or gradient centrifugation<\/span>. Mouse T-cell separation protocol is established for 2 x 107<\/sup> cells. Higher cell numbers require larger amounts of beads and MHC<\/span>.<\/p>\n

                                  \n
                                1. Centrifuge cells twice 10 minutes at 300 g at 4\u00b0C and resuspend in 10 ml buffer IS, respectively.<\/li>\n
                                2. Pass cells through enclosed 100 \u00b5m nylon mesh. This is necessary to remove cell clumps which may clog the columns.<\/li>\n
                                3. Determine cell number, take sample (before separation, approximately 1-5 x 105<\/sup> cells are required per staining) and place cells on ice.<\/li>\n
                                4. Incubate 50 \u00b5l magnetic beads and 16 \u00b5l MHC<\/span> and 80 \u00b5l buffer IS at least 45 minutes at 4\u00b0C (or over night).<\/li>\n
                                5. Place MiniMACS column in the magnetic field and prepare column by rinsing with 2 ml buffer IS.<\/li>\n
                                6. Add 1 ml buffer IS to MHC<\/span>\/magnetic beads solution and load on MS column. To wash away unbound MHC<\/span>, magnetic beads are bound and washed on a MS column.<\/li>\n
                                7. Wash with 2 ml buffer IS.<\/li>\n
                                8. Add 250 \u00b5l buffer IS and elute retained beads outside of the magnetic field into a fresh vial and firmly flush out the beads using the supplied plunger supplied with the column.<\/li>\n
                                9. Centrifuge cell suspension and resuspend in 250 \u00b5l magnetic beads\/MHC<\/span> solution. Incubate 45 minutes on ice.<\/li>\n
                                10. Add 1.5 ml buffer IS, centrifuge cell and beads mixture 10 minutes at 300 x g at 4\u00b0C and wash once by resuspending in 2 ml buffer IS and centrifuging as above. This is necessary to eliminate unbound magnetic beads which may trap cells on the column un-specifically.<\/li>\n
                                11. Resuspend in 2 ml buffer IS. Proceed to magnetic separation.<\/li>\n<\/ol>\n

                                  Magnetic separation on LS and MS columns<\/strong><\/h4>\n
                                    \n
                                  1. Place LS column in the magnetic field and prepare column by rinsing with 3 ml buffer IS.<\/li>\n
                                  2. Apply cell suspension onto the column. Allow cells to pass through and collect effluent for later analysis (.Flow-through.).<\/li>\n
                                  3. Wash column with 2 x 3 ml buffer IS.<\/li>\n
                                  4. Add 2 x 3 ml buffer IS and elute retained cells outside of the magnetic field into a fresh vial (optional: take sample for analysis).<\/li>\n
                                  5. Rinse MS column with 0.5 ml and apply cells eluted from LS column.<\/li>\n
                                  6. Wash column with 2 x 2 ml buffer IS.<\/li>\n
                                  7. Add 2 x 3 ml buffer IS and elute retained cells outside of the magnetic field into a fresh vial, take sample for analysis (eluted fraction, should contain the isolated T-cells<\/span>).<\/li>\n<\/ol>\n

                                    Magnetic separation with the autoMACS separator<\/strong><\/h4>\n
                                      \n
                                    1. Prepare and prime the autoMACS<\/li>\n
                                    2. Place tube containing the magnetically labeled cells in the autoMACS separator and choose program “PosseId”.<\/li>\n
                                    3. Collect positive fraction (outlet port “pos2”). This fraction represents the magnetically labeled antigen<\/span> specific T-cells<\/span>.<\/li>\n
                                    4. Optional: Collect negative fraction (outlet port “neg1”). This fraction represents mostly T-cells<\/span> specific for other antigens and other cell types.<\/li>\n<\/ol>\n

                                      \u00a0<\/strong>Dissociation of Strep<\/em>tamers with D-biotin<\/strong><\/h4>\n
                                        \n
                                      1. Centrifuge eluted cells and resuspend in 2 ml buffer IS containing 1 mM Dbiotin and incubate for 20 minutes.<\/li>\n
                                      2. Centrifuge cells and resuspend in 2 ml buffer IS containing 1 mM D-biotin and incubate for another 20 minutes.<\/li>\n
                                      3. Wash cells 4 x with 5 ml buffer IS.<\/li>\n<\/ol>\n

                                        Staining of T-cells with Strep<\/em>tamers<\/strong><\/h4>\n

                                        To evaluate the purity of the enriched antigen<\/span>-specific T-cells<\/span>, fractions can be analyzed by flow cytometry. Live\/dead stain can be analyzed with ethidium monazid<\/p>\n

                                        bromide (EMA), CD8<\/span>+ T-cells<\/span> can be detected using a CD8<\/span>-PE antibody, and optionally a T-cell marker like CD3 can be detected with, e.g. CD3-FITC antibody. The antigen<\/span> specific fraction of these cells can be detected by using MHC<\/span> in combination with a Strep<\/em>-Tactin-PE conjugate. When secondary staining of CD3 or CD8<\/span> is desired add the respective antibody 25 minutes after the addition of the Strep<\/em>-Tactin-PE\/MHC<\/span> I complex to the cells so that its incubation will last 20 minutes (total incubation of the Strep<\/em>-Tactin-PE\/MHC<\/span> I with the cells = 45 minutes).<\/p>\n

                                        Buffer Composition<\/strong><\/h4>\n
                                          \n
                                        • Phosphate<\/span> buffered saline (PBS): 8.06 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, pH<\/span> 7.4<\/li>\n
                                        • Buffer IS: 0.5 % BSA (w\/v), in PBS pH<\/span> 7.4<\/li>\n
                                        • Biotin stock solution isotonic 100 mM biotin\/NaCl pH<\/span> 7.4<\/li>\n<\/ul>\n

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

                                          Strep-tags are short peptides with high binding selectivity for Strep-Tactin, an engineered streptavidin. The binding affinity of e.g. Strep-tag II to Strep-Tactin (Kd = 1 \u00b5M) is nearly 100 times higher than to streptavidin. Strep-tags may be fused to recombinant proteins which allows efficient one-step purification of such fusion proteins on immobilized Strep– Tactin under […]<\/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-1816","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1816","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=1816"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1816\/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=1816"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}