GFP-CLASP1α, mCherry-CLASP2α, Tag-BFP-CLASP2α, GFP-CLASP2α, its TOG-domain truncations, point mutants, fusion proteins with the TOG domains of chTOG and coiled coil of CLIP-170, and GFP-MCAK used in the in vitro reconstitutions assays were purified from HEK293T cells using the Strep(II)-streptactin affinity purification. Cells were harvested 2 days after transfection. Cells from a 15 cm dish were lysed in 500 μl of lysis buffer (50 mM HEPES, 300 mM NaCl and 0.5% Triton X-100, pH 7.4) supplemented with protease inhibitors (Roche) on ice for 15 minutes. The supernatant obtained from the cell lysate after centrifugation at 21,000 x g for 20 minutes was incubated with 40 μl of StrepTactin Sepharose beads (GE) for 45 minutes. The beads were washed 3 times in the lysis buffer without the protease inhibitors. The protein was eluted with 40 μl of elution buffer (50 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol (DTT), 2.5 mM d-Desthiobiotin and 0.05% Triton X-100, pH 7.4). Purified proteins were snap-frozen and stored at −80°C.
Strep tactin sepharose beads
Manufactured by GE Healthcare
Strep-Tactin Sepharose beads are a solid-phase affinity chromatography material used for the purification of Strep-tagged proteins. The beads are composed of Sepharose, a cross-linked agarose matrix, with covalently attached Strep-Tactin, a modified streptavidin protein that binds to the Strep-tag II peptide sequence with high affinity. These beads provide a simple and efficient method for the capture and purification of Strep-tagged recombinant proteins from various sample sources.
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Lab products found in correlation
Protease inhibitor, by Roche (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Typhoon fla 9500 fluorescence scanner, by GE Healthcare (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
D desthiobiotin, by Merck Group (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
5 protocols using strep tactin sepharose beads
1
Affinity Purification of CLASP Proteins and Variants
2
Dynein/Dynactin Pulldown Assay with Rab45 and CRACR2a
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Pulldown of dynein/dynactin was performed as previously described (Huynh and Vale, 2017 (link)). Briefly, dynein/dynactin isolated from RPE1 cells were mixed with 20 nM purified Rab45 or CRACR2a and 15 µl preequilibrated Streptactin Sepharose beads (GE Healthcare) in 300 µl binding buffer (50 mM Hepes, pH 7.4, 20 mM NaCl, 1 mM Mg-Acetate, 10% glycerol, 0.1% NP-40, and 2 mM DTT). The mixture was incubated at 4°C for 1 h. Beads were pelleted at 1,000× relative centrifugal force for 2 min and washed four times with 500 µl binding buffer. The proteins were eluted by boiling in SDS loading buffer for 5 min and loaded onto SDS-PAGE gel. The amounts of CRACR2a and Rab45 in the pulldown samples were visualized by Coomassie staining. The amounts of dynein and dynactin were detected via Western blot with antibodies against dynein heavy chain and the p150 subunit of dynactin, respectively. In the pulldown assay shown in Fig. 2 A , 2 mM EGTA or EGTA:Ca2+ was added to the binding buffer to deplete calcium or to maintain a free calcium concentration of 2 µM. Information about antibodies used can be found in Table S1.
3
Affinity Purification of BICD2 Complex
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Pull-downs were performed as previously described (McKenney et al., 2014 (link)). 500 µl clarified porcine brain lysate in buffer A (30 mM Hepes, pH 7.4, 50 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, and 10% glycerol) was added to 80 µl of a 50% suspension of Strep-Tactin Sepharose beads (GE Healthcare) along with 100 nM of the BICD2 construct to be tested. To this, 0.1% of NP-40, 1 mM PMSF, and 5 mM DTT were added, and the mixture was incubated at 4°C for 1 h. The beads were then pelleted and washed four times with buffer A containing 0.1% NP-40 and 5 mM DTT. The proteins were then eluted using SDS loading buffer and resolved on an acrylamide gel (Invitrogen). For experiments in which Rab6a was supplemented, the appropriate amount of recombinant Rab6a protein was added before the 4°C incubation step and processed in the same fashion.
4
Affinity Purification of CPSF Complexes
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For pull-down assays from Sf9 cells expressing CPSF subunits, the cells were resuspended in 20 mM Tris-Cl pH 7.5, 200 mM NaCl, 10% glycerol and 0.05% Tween20, and lysed by sonication. The clarified lysate was incubated with 600 μl of NiNTA resin (Qiagen). After washing, the protein was eluted and incubated directly with 30 μl Streptactin sepharose beads (GE Healthcare). The beads washed three times with 1 ml of resuspension buffer and the protein eluted with SDS-PAGE loading buffer at room temperature. The eluted samples and the input lysate were loaded on SDS-PAGE with no prior boiling to avoid GFP denaturation, visualized on a Typhoon FLA9500 fluorescence scanner (GE Healthcare) and subsequently stained with Coomassie brilliant blue R250.
For pull-down assays with purified proteins, 3 μg of GST-Fip1CD was immobilized on glutathione sepharose four fast flow beads (GE Healthcare) and incubated with 40 μg of MBP-CPSF30. The beads were washed three times with 500 μl of buffer containing 20 mM Tris-Cl pH 7.5, 150 mM NaCl and 0.05% Tween20, the sample eluted with SDS-PAGE loading buffer and analyzed by SDS-PAGE.
For pull-down assays with purified proteins, 3 μg of GST-Fip1CD was immobilized on glutathione sepharose four fast flow beads (GE Healthcare) and incubated with 40 μg of MBP-CPSF30. The beads were washed three times with 500 μl of buffer containing 20 mM Tris-Cl pH 7.5, 150 mM NaCl and 0.05% Tween20, the sample eluted with SDS-PAGE loading buffer and analyzed by SDS-PAGE.
5
Purification of PRPH2-ROM1 Heterodimer
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N-terminal 6x-His–tagged PRPH2C150S and N-terminal Strep3-GFP–tagged ROM1 were diluted with dummy plasmid expressing the tripeptide methionine-glycine-serine (MGS) and transiently transfected into 2 liters of HEK293T cells (provided by U-Protein Express BV). Cells were grown at 37°C and harvested by centrifugation (10 min, 1000g at 4°C) after 96 hours. The harvested cell pellets were lysed and solubilized for 2 hours at 4°C with gentle agitation in buffer A comprising 50 mM tris-HCl, 150 mM NaCl at pH 7.5, EDTA-free complete protease inhibitor tablet (Roche), 1% (w/v) DDM, and 0.1% (w/v) CHS. Samples were ultracentrifuged (40 min, 40,000 rpm at 4°C), and supernatants were then incubated with Strep-Tactin Sepharose beads (GE Healthcare Life Sciences) for 2 hours at 4°C in a buffer containing 0.025% (w/v) DDM/CHS. Proteins were eluted in 2.5 mM d-desthiobiotin (Sigma-Aldrich) and injected onto a Superdex 200 Increase 10/300 GL (GE Healthcare Life Sciences) SEC column preequilibrated with buffer B comprising 25 mM tris-HCl, 150 mM NaCl at pH 7.5, and 0.025% (w/v) DDM/CHS. Fractions containing PRPH2-ROM1 heterodimer were pooled and incubated with an excess of Nanobody19 and concentrated in a 100-kDa concentration device (Amicon) to ~9 mg/ml.
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