{"id":124,"date":"2025-06-10T16:41:51","date_gmt":"2025-06-10T23:41:51","guid":{"rendered":"https:\/\/test-inside.ewu.edu\/jashley6\/?page_id=124"},"modified":"2025-06-11T10:37:07","modified_gmt":"2025-06-11T17:37:07","slug":"indirect-western-blotting-with-biotinylated-antibodies-with-no-stain-reagent","status":"publish","type":"page","link":"https:\/\/test-inside.ewu.edu\/jashley6\/indirect-western-blotting-with-biotinylated-antibodies-with-no-stain-reagent\/","title":{"rendered":"Indirect Western Blotting with biotinylated antibodies with No-Stain Reagent"},"content":{"rendered":"\n

<\/h1>\n\n\n\n

                This protocol has more steps and requires more reagents than a conventional Western blot, but it greatly enhances signal from proteins with low abundance. For more information see \u201cImproving the Sensitivity of Traditional Western blotting via Streptavidin containing Poly Horseradish Peroxidase (PolyHRP)<\/a>\u201d Manish Mishra et al.<\/p>\n\n\n\n

Equipment<\/h2>\n\n\n\n
Mini Gel Tank (ThermoFisher, A25977)<\/td>Oscillating platform in refrigerator<\/td><\/tr>
Gel spatula<\/td>Glass trays<\/td><\/tr>
Trans-Blot Turbo Transfer System (Bio-Rad)<\/td>Plastic lidded boxes<\/td><\/tr>
Gel roller<\/td>Incubator<\/td><\/tr>
Plastic forceps<\/td>Tube rotator<\/td><\/tr>
Rocking platform<\/td>Digital imager<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

Materials<\/h2>\n\n\n\n
    \n
  • Protein sample<\/li>\n\n\n\n
  • 4X Bolt\u2122 LDS Sample Buffer (ThermoFisher, B0008)<\/li>\n\n\n\n
  • 10X Bolt\u2122 Sample Reducing Agent (ThermoFisher, B0009)<\/li>\n\n\n\n
  • PageRuler\u2122 Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher, 26616)<\/li>\n\n\n\n
  • Bolt\u2122 Bis-Tris Plus Mini Protein Gels, 4-12% (ThermoFisher, NW04122BOX)<\/li>\n\n\n\n
  • 20X Bolt\u2122 MES SDS Running Buffer (ThermoFisher, B0002)<\/li>\n\n\n\n
  • Bolt\u2122 Antioxidant (ThermoFisher, BT0005)<\/li>\n\n\n\n
  • No-Stain\u2122 Protein Labeling Reagent (ThermoFisher, A44717)<\/li>\n\n\n\n
  • Trans-Blot Turbo RTA Mini 0.45 \u00b5m LF PVDF Transfer Kit (Bio-Rad, #1704274)<\/li>\n\n\n\n
  • Acetone<\/li>\n\n\n\n
  • Methanol<\/li>\n\n\n\n
  • 20X Tris-buffered saline with tween-20 (TBS-T) (ThermoFisher, 28360)<\/li>\n\n\n\n
  • Non-fat dry milk (Cell Signaling Technology, #9999)<\/li>\n\n\n\n
  • Molecular Probes\u2122 D-Biotin (ThermoFisher, B1595)<\/li>\n\n\n\n
  • Streptavidin (MedChem Express, HY-P3152-10mg)<\/li>\n\n\n\n
  • Bovine serum albumen (Cell Signaling Technology, #9998)<\/li>\n\n\n\n
  • Primary antibodies<\/li>\n\n\n\n
  • Biotinylated secondary antibodies (e.g. Goat anti Rabbit IgG (H+L) Secondary Antibody, Biotin XX, Invitrogen \u2013 ThermoFisher, B2770)<\/li>\n\n\n\n
  • Pierce\u2122 Streptavidin Poly-HRP (ThermoFisher, 21140)<\/li>\n\n\n\n
  • Plastic sheet protector<\/li>\n\n\n\n
  • Chemiluminescent substrate (Cell Signaling Technology, SignalFire\u2122 ECL Reagent #6883 or SignalFire\u2122 Elite ECL Reagent #12757)<\/li>\n<\/ul>\n\n\n\n

    Method<\/h2>\n\n\n\n

    Day 1<\/h3>\n\n\n\n
      \n
    1. Prepare protein samples (10-40\u00b5g) in LDS sample buffer with reducing agent in a volume of 30\u00b5L or less.<\/li>\n\n\n\n
    2. Set up gel will 1X running buffer with antioxidant.<\/li>\n\n\n\n
    3. Load samples and run gel at 200V for 20-60min.\n
        \n
      1. During gel run, preheat incubator to 56\u00b0C.<\/li>\n<\/ol>\n<\/li>\n\n\n\n
      2. Transfer proteins using the standard Trans-Blot Turbo protocol
        1. 25V, 1A, 30 min<\/li><\/ol>
          1. During transfer prepare cold acetone in glass trays on ice<\/li><\/ol>\n
              \n
            1. During transfer prepare 5% milk and 5% BSA solutions in 1X TBS-T\n
                \n
              1. Each membrane requires 10mL milk and 50mL BSA<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/li>\n\n\n\n
              2. No-Stain labeling and imaging of membrane
                1. Prepare staining solution
                  1. 9.5mL deionized water<\/li><\/ol>
                    1. 0.5mL 20X No-Staining Labeling Buffer<\/li><\/ol>
                      1. 20\u00b5L No-Stain Activator<\/li><\/ol>
                        1. 20\u00b5L No-Stain Derivatizer<\/li><\/ol><\/li><\/ol>
                          1. Place 10mL staining solution into a glass tray and add membrane with transferred proteins<\/li><\/ol>
                            1. Rock slowly (~60rpm) for 10 minutes at room temperature.<\/li><\/ol>
                              1. Discard labeling solution<\/li><\/ol>
                                1. Wash membrane with deionized water 3\u00d72 minutes with slow rocking.<\/li><\/ol>
                                  1. Discard final wash solution and replace with deionized water<\/li><\/ol>\n
                                      \n
                                    1. Place membrane in imager and illuminate with blue light; image using the orange DNA filter<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                    2. Place membrane in cold acetone and incubate for 30 minutes with gentle rocking<\/li>\n\n\n\n
                                    3. Pour off acetone in beaker in fume hood<\/li>\n\n\n\n
                                    4. Place trays in preheated incubator and incubate for 15-30 minutes until membranes are completely dry\n
                                        \n
                                      1. After first 1-2 minutes, open the door to vent acetone vapor<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                      2. Rinse membranes quickly with methanol and place in small plastic lidded boxes with 10mL 5% milk<\/li>\n\n\n\n
                                      3. Incubate membrane at room temperature for 60 minutes with gentle rocking.\n
                                          \n
                                        1. During incubation prepare 10mL 0.1 mg\/mL streptavidin and 10mL 0.5mg\/mL biotin blocking solutions in 5% BSA.<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                        2. Wash membrane 3 x 5 minutes with vigorous rocking<\/li>\n\n\n\n
                                        3. Incubate membrane in 10mL streptavidin blocking solution at room temperature for 15 minutes with gentle rocking<\/li>\n\n\n\n
                                        4. Wash membrane 3 x 5 minutes with vigorous rocking<\/li>\n\n\n\n
                                        5. Incubate membrane in 10mL biotin blocking solution at room temperature for 60 minutes with gentle rocking\n
                                            \n
                                          1. During incubation, prepare primary antibody dilution (1:1000 \u2013 1:5000) in 10mL 5% BSA<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                          2. Wash membrane 3 x 5 minutes with vigorous rocking<\/li>\n\n\n\n
                                          3. Incubate membrane in primary antibody solution at 4\u00b0C overnight with gentle oscillation.<\/li>\n<\/ol>\n\n\n\n

                                            Day 2<\/h3>\n\n\n\n
                                              \n
                                            1. Wash membrane 3 x 5 minutes with vigorous rocking\n
                                                \n
                                              1. During washes, prepare secondary antibody dilution (1:5000 \u2013 1:10000) in 10mL 5% BSA<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                              2. Incubate membrane in secondary antibody solution at room temperature for 60 minutes with gentle rocking\n
                                                  \n
                                                1. During incubation prepare streptavidin-PolyHRP dilution (50 \u2013 100ng\/mL) in 10mL 5% BSA<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                                2. Wash membrane 3 x 5 minutes with vigorous rocking<\/li>\n\n\n\n
                                                3. Incubate membrane in streptavidin-PolyHRP solution at room temperature for 60 minutes with gentle rocking<\/li>\n\n\n\n
                                                4. Wash membrane 3 x 5 minutes with vigorous rocking<\/li>\n\n\n\n
                                                5. Transfer membrane to small plastic box<\/li>\n\n\n\n
                                                6. Prepare chemiluminescent substrate (0.5mL per membrane) and apply to membrane.<\/li>\n\n\n\n
                                                7. Manually rock membrane for 30 seconds to coat with substrate<\/li>\n\n\n\n
                                                8. Transfer membrane to plastic sheet protector and roll off bubbles and excess.<\/li>\n\n\n\n
                                                9. Take a 25-image stack using the imager with no filter and 2X to 4X camera binning\n
                                                    \n
                                                  1. Start with a 5-second exposure time and adjust up or down as necessary<\/li>\n<\/ol>\n<\/li>\n\n\n\n
                                                  2. Before removing the membrane capture a white light illuminated image of the membrane (you\u2019ll need to a low exposure and low brightness setting to avoid washout) for later overlay of the ladder onto the blot image.\n
                                                      \n
                                                    1. Optional \u2013 to have a color image of the ladder for future reference, take 3 images with white illumination through red, green, and blue filters, false color them to their associated filter, and digitally merge them.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

                                                                      This protocol has more steps and requires more reagents than a conventional Western blot, but it greatly enhances signal from proteins with low abundance. For more information see \u201cImproving the Sensitivity of Traditional Western blotting via Streptavidin containing Poly Horseradish Peroxidase (PolyHRP)\u201d Manish Mishra et al. Equipment Mini Gel Tank (ThermoFisher, A25977) Oscillating platform … Read more<\/a><\/p>\n","protected":false},"author":66,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"wpo365_audiences":[],"wpo365_private":false,"footnotes":""},"class_list":["post-124","page","type-page","status-publish"],"_links":{"self":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/124"}],"collection":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/users\/66"}],"replies":[{"embeddable":true,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/comments?post=124"}],"version-history":[{"count":3,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/124\/revisions"}],"predecessor-version":[{"id":143,"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/pages\/124\/revisions\/143"}],"wp:attachment":[{"href":"https:\/\/test-inside.ewu.edu\/jashley6\/wp-json\/wp\/v2\/media?parent=124"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}