G-actin (1 mg/ml) Cytostebu-bio, Cytoskeleton) was incubated on ice for 1 hour and centrifuged at 4 °C for 20 min at 14000 g. HiLyteFluor 647-tubulin was purchased from Cytoskeleton and HyLight647-MT seeds were made as described in Mohan et al. 201341 (link) and stored at −80 °C. For the experiment seeds were quickly transferred into a 37 °C water bath, incubated for 20 min and kept in the dark at RT for 1–2 hrs. Labeled MTs were diluted 1:40 in PEM80 (80 mM PIPES, pH 6.9, 2 mM MgCl2, 1 mM EGTA) containing 10 µM of taxol (Sigma). 8 µl Drebrin or DrebrinS142D (50 ng/µl), 1 µl EB3 (100 ng/µl), 1 µl ATP (5 mM), 1 µl GTP (20 mM), 2 µl taxol (200 µM), 2 µl 10x GT-buffer (800 mM PIPES pH 7.0, 20 mM MgCl2, 5 mM EGTA), 2 µl Alexa-Fluor®488 labeled phalloidin (1:25 dilution), 2 µl G-actin (1 mg/ml; tebu-bio, Cytoskeleton), 1 µl MTs were added to a microtube, mixed, applied onto a poly-L-lysine covered chamber slide (Ibidi, Munich) and analyzed by TIRF-microscopy. TIRFM was performed on Visiscope Imaging system described above.
G actin
Manufactured by Cytoskeleton
Sourced in United States
G-actin is a globular protein that is a monomeric subunit of actin filaments. It plays a crucial role in the formation and dynamics of the cytoskeleton in eukaryotic cells.
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Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Cary eclipse fluorimeter, by Agilent Technologies (2 mentions)
F actin, by Cytoskeleton (1 mentions)
Taxol, by Cytoskeleton (1 mentions)
Poly l lysine covered chamber slide, by Ibidi (1 mentions)
Taxol, by Merck Group (1 mentions)
Tabletop centrifuge, by Beckman Coulter (1 mentions)
Optima le 80k, by Beckman Coulter (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Arp2 3 complex, by Cytoskeleton (1 mentions)
Infinite m1000 plate reader, by Tecan (1 mentions)
Instantblue protein stain, by Abcam (1 mentions)
Tla 120.1 rotor, by Beckman Coulter (1 mentions)
Envision 2103, by PerkinElmer (1 mentions)
17 protocols using g actin
1
Visualizing Actin-Microtubule Interactions
2
Baculoviral Expression of Dynamin Variants
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Human Dyn1, rat Dyn2, human Dyn1∆PRD, and rat Dyn2∆PRD were expressed using Bac-to-Bac baculovirus expression systems (ThermoFisher Scientific) in Sf21 insect cells10 (link). Recombinant human gelsolin (Gsn) (gift from Fumihiko Nakamura, Brigham and Women’s Hospital); purified non-labeled G-actin, pyrene labeled G-actin (Cytoskeleton); human uPAR protein (R&D Systems); Rhodamine phalloidin (ThermoFisher Scientific).
3
Actin Polymerization Assay with GAS2L1
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Rabbit skeletal muscle G-actin (>99% purity; Cytoskeleton Inc.) was diluted in G-buffer containing 0.2 mM ATP and 0.5 mM dithiothreitol, and GAS2L1 proteins (His6-FLAG tagged) were diluted in actin polymerization buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl2, and 1 mM ATP); the G-actin and GAS2L1 proteins were then clarified by centrifugation at 150,000 g at 4°C for 30 min. Next, F-actin was polymerized in the actin polymerization buffer for 1 h at room temperature, and the F-actin polymerized from 5 µM G-actin was incubated with the GAS2L1 proteins (5 µM) in the polymerization buffer for 30 min at room temperature. Last, the samples were centrifuged at 150,000 g for 30 min at 24°C, and then both the supernatants and the pellets were analyzed on immunoblots.
4
Actin Polymerization Analysis in Cells
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The actin polymerization state in shCRMP1-transduced cells was determined by an F-actin (filamentous form)/G-actin (globular form) in vivo assay (Cytoskeleton, Inc., Denver, CO, USA). Briefly, cells were lysed in an F-actin stabilizing buffer (50 mm PIPES, pH 6.9, 50 mm NaCl, 5 mm MgCl2, 5 mm EGTA, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween-20, 0.1% 2-mercaptoethanol, 0.001% Antifoam C and protease inhibitors). The cell lysates were centrifuged at 106 g to separate the F-actin (pellets) from G-actin (supernatants). F-actin in pellet fraction was then depolymerized in water containing 8 m urea. Actin in each fraction was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and quantitated by immunoblotting with an anti-actin antibody. The immunosignal intensities were quantified using gel densitometry software (ImageJ, NIH, Bethesda, MD, USA) and the ratio of F-actin/G-actin was determined.
5
Far-Western Blotting Assay for Protein-Protein Interactions
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Far-Western blotting was performed as described previously (Wang et al., 2011 (link)). Equal amounts of purified GST and GST fusion proteins were resolved by SDS-PAGE and transferred to PVDF membranes. Then the membranes were blocked in basic buffer (20 mM Hepes, pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, and 0.1% Igepal CA-630) containing 10% nonfat dry milk overnight at 4°C and then incubated in interaction buffer (basic buffer with 1.5% nonfat dry milk) containing 2 µg/ml of purified His-MTCBP-1 protein or 4 µM of F-actin for 1 h at room temperature. The F-actin was polymerized from G-actin (Cytoskeleton, Inc.) based on the manufacturer’s protocol. The membranes were washed three times with PBS-T and probed with antibodies to MTCBP-1, His epitope tag (Cell Signaling), or actin. The membrane was then stripped and probed with GST to confirm equal loading of the GST proteins. For experiments testing if MTCBP-1 disrupts the preestablished interaction between GST fusion proteins and F-actin, the membranes containing GST fusion proteins were incubated with interaction buffer containing 0.35 µM MTCBP-1 or not for 2 h at room temperature, following the actin incubation. The subsequent steps were similar to the standard far-Western blotting assays.
6
Actin-Based Protein Interaction Assay
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G-actin (rabbit skeletal muscle actin protein, >99% pure; Cytoskeleton, Inc.) was prepared according to the manufacturer’s protocol with G buffer (2 mM Tris, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT). All purified proteins including GST, GST-MT1-MMP-CT, and HIS-MTCBP-1 were dialyzed in G buffer after purification and subjected to centrifugation (100,000 g for 20 min at 4°C) to remove insoluble proteins. The indicated amount of purified proteins and G-actin were added into each individual reaction in a total volume of 200 µl that was equilibrated with G buffer. Subsequently, 20 µl of F buffer (20 mM Tris, pH 7.5, 2 mM CaCl2, 1 M KCl, 20 mM MgCl2, 10 mM ATP, and 5 mM DTT) was added to each reaction and incubated for 1 h before pelleting (100,000 g for 1 h at 25°C) in a Beckman table-top centrifuge. Pellets and supernatants were separated and brought up to equal volume and 40 µl of the samples were resolved by SDS-PAGE gels and immunoblotted.
7
Actin-Protein Binding Kinetics Assay
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Skeletal muscle G-Actin (Cytoskeleton, Denver, USA) was polymerized (7 µM, final concentration) and incubated at room temperature with varying concentrations of either the WT or the mutant protein. Above mix (final volume 100 µl) was centrifuged at 100,000 g for 30 min (sw55Ti rotor, Beckman Optima LE80K) and pellets were solubilized in 30 µl SDS-PAGE loading buffer. Half of this was boiled and subjected to SDS-PAGE electrophoresis and was stained with coomassie brilliant blue. Intensities of the individual bands were determined using the QuantityOne software on Biorad Gel Doc XR. These intensity values were corrected for the differential staining of proteins with Coomassie Blue as described before [23] (link), [24] (link). Ratios of the corrected intensities were used to determine the fraction bound of actin and free protein (either the WT or mutant). Fraction bound of F-actin was plotted against free protein to obtain the binding curve.
8
Actin Polymerization Assay with SYMREM1 and mDia1
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Purified SYMREM1 (10 µM or 30 µM) or mDia1-ct (10 µM) proteins were incubated with 10 µM G-actin (Cytoskeleton Inc.) for 1 h at RT in an actin-polymerization buffer (F-buffer: 5 mM Tris-HCl pH 7.5, 100 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EGTA, 0.2 mM ATP and 0.5 mM DTT). After ultracentrifugation at 80,000 × g for 30 min the supernatant was removed, as the non-pelleted fraction. The remaining pellet was then washed one time with F-buffer and subsequently resuspended in an equal volume compared to the supernatant. Laemmli buffer was added to all lysates, boiled at 96 °C for 10 min and conducted to SDS-page analysis.
9
Purification and Polymerization of Tubulin and Actin
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Tubulin was purified by two cycles of polymerization and depolymerization from porcine brain as described previously (45 (link)). Taxol-stabilized MTs were polymerized by the stepwise addition of taxol (45 (link)). Both tubulin and MTs were stored at −80 °C. MTs were thawed rapidly at 37 °C immediately before use.
Globular actin (G-actin) was purified from rabbit muscle acetone powder (Pel-Freez Biologicals) by a cycle of polymerization and depolymerization as described previously (46 (link)). Purified G-actin was stored in a dialysis bag in calcium buffer G (2 mM Tris–HCl, 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2, 1 mM sodium azide, pH 8), and the buffer was refreshed weekly. To polymerize F-actin, G-actin was first converted to MG-actin with 5 mM MgCl2 and 0.2 mM EGTA buffer for 5 min at room temperature. Then, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole–HCl (pH 7) buffer was added for 1 h at room temperature to polymerize F-actin. The same process was performed with calcium buffer G to generate a complementary buffer for reaction without any F-actin as a negative control (Fig. 1 B). For Fig. S1 , commercial G-actin (catalog no.: AKL99-B; Cytoskeleton, Inc) was used. The G-actin was reconstituted according to the protocol from Cytoskeleton, Inc before polymerization. The F-actin polymerization steps were the same as described above.
Globular actin (G-actin) was purified from rabbit muscle acetone powder (Pel-Freez Biologicals) by a cycle of polymerization and depolymerization as described previously (46 (link)). Purified G-actin was stored in a dialysis bag in calcium buffer G (2 mM Tris–HCl, 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2, 1 mM sodium azide, pH 8), and the buffer was refreshed weekly. To polymerize F-actin, G-actin was first converted to MG-actin with 5 mM MgCl2 and 0.2 mM EGTA buffer for 5 min at room temperature. Then, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM imidazole–HCl (pH 7) buffer was added for 1 h at room temperature to polymerize F-actin. The same process was performed with calcium buffer G to generate a complementary buffer for reaction without any F-actin as a negative control (
10
Gelsolin Severing Activity on Actin
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Pyrene-labeled rabbit skeletal muscle globular actin (G-actin; Cytoskeleton, Inc. (Denver, CO, USA)) was used to evaluate the severing activity of the pathological variants of gelsolin. Preparation of the solutions, actin manipulation and conversion of G-actin to filamentous actin (F-actin) were reported elsewhere [52] (link). Measurements were made at 20 °C with a Cary Eclipse fluorimeter (Agilent Technologies, USA), with the following settings: excitation and emission wavelength/slit, respectively, 365/5 and 407/5 nm; averaging time 0.1 s. A 3 mL cuvette was used, with a stirring bar for continuous agitation of the reaction mixture; 400 μL of a 4 μM F-actin solution were incubated in the cuvette until stabilization of the fluorescence signal, then 2 μL of 50 μM GSN were added (0.25 μM final concentration). Once the signal was stable again, the severing reaction was started by adding 2 μL of a 1 M CaCl2 solution (final free Ca2+ concentration > 1 mM). Data were normalized based on initial and end-point fluorescence measured in the presence of Ca2+. For the Ca2+-free assays, measurement is started on addition of the proteins and the depolymerization rate is calculated by linear fitting over 3 min.
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