The assay was performed as previously described with small modifications (Espeut et al., 2012 (link)). In brief, decoration of Spc105 proteins on polystyrene beads was achieved using streptavidin-biotin system. 100 nm each of WT and mutant Spc105 proteins was incubated with 10 µl of 0.1% (wt/vol) streptavidin polystyrene beads (Spherotech) conjugated with anti-penta-his biotin antibody (Qiagen) for 90 min at 4°C. Before imaging, the beads were sonicated for 3 min in a water bath containing ice cubes. Subsequently, the beads were introduced inside the flow chamber coated with taxol-stabilized microtubules. The images were acquired using a TIRF microscope.
Streptavidin polystyrene beads
Manufactured by Spherotech
Streptavidin polystyrene beads are uniform, monodisperse beads composed of polystyrene and coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, enabling the beads to be used as a solid support for biotinylated molecules.
Automatically generated - may contain errors
Lab products found in correlation
Polystyrene beads, by Spherotech (2 mentions)
Carboxylated polystyrene beads, by Polysciences (1 mentions)
Biotinylated bsa, by Merck Group (1 mentions)
Sulfo smcc crosslinker, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti digoxigenin fab fragments, by Roche (1 mentions)
Complete mini cocktail, by Roche (1 mentions)
Phospho akt ser473 ab, by Cell Signaling Technology (1 mentions)
Phospho thr202 tyr204 clone d13.14.4e, by Cell Signaling Technology (1 mentions)
Gapdh clone d16h11, by Cell Signaling Technology (1 mentions)
4 protocols using streptavidin polystyrene beads
1
Spc105 Protein Bead Decoration Assay
2
Crosslinker-Mediated Biomolecular Conjugation
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Sulfo-SMCC crosslinker (Thermo Scientific—22622), 1,010 bp DNA functionalized with appropriate end groups (primers specified in the following methods), 1.25 μm streptavidin polystyrene beads (Spherotech—SVP-10-5), 0.75 μm polystyrene beads (Spherotech—PP-08-10), 1.0 μm carboxylated polystyrene beads (Polysciences—08266), biotinylated BSA (5 mg ml−1, received as a gift from Ted Feldman at Harvard University), BSA (Sigma—A3059), 50 mM acetate buffer (pH 4.9), PBS (1 × , pH 7.4), Whatman Grade 1 Filter Paper (Sigma—WHA1001110), Trichoderma reesei cellulase mixture (Sigma—C2730) and deionized (DI) water were used.
3
DNA Origami Tether Experiments
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Constant-trap-position experiments have been performed on a dual optical tweezer (C-TrapTM from Lumicks, The Netherland). Opposite helical domains of the DNA origami structure were linked to two double-stranded 3 kbp-long DNA handles, functionalized at the ends either with digoxygenin or biotin. Digoxygenin- and biotin-functionalized handles were obtained by PCR amplification of the pET-28a vector (5369 bp). DNA handles, bridging the DNA origami to the beads, contained a central PEG9 spacer flanked by two ca. 30 nt-long stretches, one partially complementary to the M13 scaffold and the other partially complementary to the tether. For the tweezers experiment, DNA origami-handles constructs were tethered between 1.76 µm streptavidin polystyrene beads (Spherotech, Inc) and 1 µm polystyrene beads (Spherotech, Inc), previously covalently functionalized with anti-digoxigenin Fab fragments (Roche). Measurements were carried out at room temperature in 1× TEMg buffer with the addition of an oxygen scavenger system (26 U/ml glucose oxidase, 17,000 U/ml catalase).
4
Signaling Assays in Cytotoxic T Cells
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In total, 0.2–1 × 106 CTLs were lysed using cold cell lysis buffer containing 50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, and 0.25% sodium deoxycholate. Suppression of talin 1 was confirmed using an anti-talin 1 antibody (clone 8D4, Abcam). Talin head domain GFP fusion was detected using a polyclonal antibody against GFP (Invitrogen). Actin (clone AC-15, Sigma) or GAPDH (clone D16H11, Cell Signaling Technology) served as loading controls. For signaling assays, serum and IL-2 starved OT-1 CTLs were incubated with streptavidin polystyrene beads (Spherotech) coated with H-2Kb-OVA and ICAM-1 at a 1:1 ratio for various times at 37 °C and immediately lysed in 2× cold lysis buffer containing phosphatase inhibitors (1 mM NaF and 0.1 mM Na3VO4) and protease inhibitors (cOmplete mini cocktail, EDTA-free, Roche). Activation of PI3K and MAP kinase signaling was assessed by immunoblot for pAkt (Phospho-Akt (Ser473) Ab; Cell Signaling Technology) and pErk1/2 (Phospho-Thr202/ Tyr204; clone D13.14.4E; Cell Signaling Technology). Additional information about antibodies may be found in Supplementary Table 1 . Uncropped images of blots are provided in Supplementary Fig. 12 .
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