tubulin buffer  (Cytoskeleton Inc)

Journal: Nature Communications

Article Title: Disruption of microtubules with low intensity ultrasound rescues hair follicle damage by paclitaxel in mouse models

doi: 10.1038/s41467-026-75335-8

Figure Lengend Snippet: A Illustration of experimental design: the 3-week-old mice were injected i.p. with 12 mg/Kg (body weight) paclitaxel (PTX). After 4 hours, the mice were exposed to ultrasound (1 W/cm 2 , 45 kHz) for 5 min, and euthanized, biopsied, and assayed 10 min later. The experiments were repeated 3 times with similar results, and 6 mice in each group ( n = 6) were used. B Skin biopsies were analyzed by histology technique, and the hair follicle sections stained with alpha-Tubulin were shown for examples of cross-section (upper panel) and sagittal section (lower panel). C Areas of the hair follicles were shown in high magnification to visualize microtubules stained with anti-alpha-tubulin. Images were processed using NeuronJ from ImageJ, employing vectorization and skeletonization techniques to measure microtubule length. The lower panel shows images of microtubule vectorization. D The length of microtubule filaments was measured using an image analytical program. “*” p < 0.01; “****” p < 0.001. E Examples were shown for mitotic figures, and shown in higher magnification in the lower panel. F Quantitation of normal, abnormal, and disrupted mitotic figures in cells of the hair follicles in the skin biopsies.

Article Snippet: Briefly, one aliquot of 488 tubulin (Cytoskeleton, TL488M) was resuspended to 5 mg/mL (45.5 μM) in 4.0 μl of general tubulin buffer (Cytoskeleton, BST01) supplemented with 0.01 volumes of 100 mM GTP (Cytoskeleton, BST06) and 1 μl of microtubules cushion buffer (Cytoskeleton, BST05) to facilitate polymerization.

Techniques: Injection, Staining, Quantitation Assay

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The endocytic fission protein EHD1 interacts with tubulin and regulates microtubule function

doi: 10.1016/j.bbamcr.2026.120120

Figure Lengend Snippet: EHD1-tubulin interactions are unlikely direct and are not mediated by MICAL-L1. (A) In-vitro binding was conducted by incubating 2 μg of purified porcine αβ-tubulin heterodimers with either 8 μg of GST alone (control) or GST-EH-1 purified on glutathione resin for 3 h at 4°C. Bead bound fractions were subjected to SDS-PAGE and then transferred to nitrocellulose and blotted with anti-α-tubulin antibody (A, top) and anti-GST antibody (A, bottom). B-D, HeLa cells were treated with siRNA oligonucleotides targeting MICAL-L1 for 72 h. (B) Immunoblotting was done to validate depletion of MICAL-L1. Lysates from mock and MICAL-L1 siRNA treated cells were subjected to coimmunoprecipitation by anti-EHD1 antibody or immunoprecipitation with beads only (control). The bead-bound fractions were separated using SDS-PAGE, transferred to nitrocellulose and then immunoblotted with anti-TUBB3 antibody (C,D) for EHD1 coIP and lysate lanes, and with anti-EHD1 antibody to confirm immunoprecipitation (EHD1 IP lane). Gels denoting TUBB3 in the coIP lane are taken from higher exposures. Depicted blots are representatives taken from three independent experiments.

Article Snippet: Purified porcine αβ-tubulin heterodimers (Cytoskeleton, Inc. #T240A) reconstituted in general tubulin buffer (80 mM PIPES pH 6.9, 2 Mm MgCl 2 , 0.5 mM EGTA) were used as target proteins.

Techniques: In Vitro, Binding Assay, Purification, Control, SDS Page, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

doi: 10.1038/s41467-026-69244-z

Figure Lengend Snippet: a Schematic depicting the formation of phase-separated protein condensates and their time-dependent transition to form amyloid fibrils. b Domain diagram of SynTag-Tau consisting of full-length Tau (2N4R isoform) with an N-terminal synthetic prionogenic tag (SynTag; sequence provided in green). c Alphafold3 predicted structure of SynTag-Tau. The green arrow marks the location of the tag. d Time-lapse imaging of Atto488-labeled SynTag-Tau condensates. Grey arrows indicate the attachment of fibrils emerging from one condensate to neighboring condensates. Also, see Supplementary Video . e Schematic of free energy landscape illustrating the relative stability of monomeric Tau, liquid-like Tau condensates, and amyloid fibrils, either with or without SynTag. f Second harmonic generation (SHG) microscopy shows the absence of molecular ordering in nascent SynTag-Tau condensates (indicated by dashed circles; sample age = 2 h) and the presence of spatial ordering in fibrils (sample age = 10 h). The corresponding quantification is also shown. g Representative Thioflavin T (ThT) fluorescence line profiles from a nascent SynTag-Tau condensate (indicated by a dashed circle; sample age = 2 h) and fibrils (sample age = 10 h). The contrast was adjusted independently in order to better visualize the nascent condensate that is very dim. h Optical tweezer-controlled fusion of nascent SynTag-Tau condensates (sample age = 30 mins, indicated as the 0-h time point) and condensates at 2 h of age. i FRAP measurements of SynTag-Tau condensates at various time points as indicated. The individual dots represent the mean value at each time point, based on data from at least three independent replicates (sample size: 2 h, 4 droplets; 4 h and 6 h, 5 droplets each). Intensity error bars were plotted based on the standard error of the mean (S.E.M.) at each time point and are represented as the shaded regions. j Peak fitting of averaged amide I spectra obtained from broadband coherent anti-Stokes Raman (BCARS) hyperspectral imaging shows the structural profile of SynTag-Tau in condensates at 2 h (nascent condensates) and at 6 h (aged condensates), as well as fibrils formed at 10 h of age. Spectra from multiple samples within each independent experiment (n ≥ 6 per experiment) were averaged prior to analysis. Data are presented as mean values with S.E.M. Each experiment was independently repeated at least three times. Source data are provided as a Source Data file.

Article Snippet: Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl 2 , and 0.5 mM EGTA, supplemented with 2 mM DTT.

Techniques: Sequencing, Imaging, Labeling, Microscopy, Fluorescence

Journal: Nature Communications

Article Title: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

doi: 10.1038/s41467-026-69244-z

Figure Lengend Snippet: a Schematic of tubulin recruitment and MT polymerization in Tau condensates. b Nascent SynTag-Tau condensates (age = 2 h) in the presence of 50 nM HiLyte647-labeled tubulin show enrichment of tubulin in the dense phase. Corresponding line profiles are shown below. c Aged SynTag-Tau condensates (age = 4 h) with the addition of 50 nM HiLyte647-labeled tubulin show altered tubulin partitioning to the condensate interface. Corresponding line profiles are shown below. Display ranges in images of (b) and (c) were adjusted independently for better clarity. d Condensate age-dependent MT polymerization assay in SynTag-Tau condensates. e MT surface coverage plot corresponding to panel (d). Horizontal lines at each time point represent the median value. The individual data points represent measurements based on three independent replicates. Sample size: 1 hr, 30 datapoints; 2.5 hr, 24 datapoints; 5 hr, 23 datapoints; 8 hr, 24 datapoints; 12 hr, 16 datapoints; 16 hr, 14 datapoints. f Frequency-domain (FD) FLIM map of SynTag-Tau condensates at various time points. g Representative fluorescence lifetime distributions at 1 h and 5 h time points (since sample preparation). The points are the data, and the lines are Gaussian fittings with the corresponding goodness of fit (R 2 ) indicated. h FD-FLIM map of aged SynTag-Tau condensates with emergent amyloid fibrils (sample age = 8 h). i Schematic of condensate physical aging-induced interfacial resistance in SynTag-Tau condensates, and its effect on tubulin partitioning and MT assembly. Each of these experiments was independently repeated at least three times. Source data are provided as a Source Data file.

Article Snippet: Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl 2 , and 0.5 mM EGTA, supplemented with 2 mM DTT.

Techniques: Labeling, Polymerization Assay, Fluorescence, Sample Prep

Journal: Nature Communications

Article Title: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

doi: 10.1038/s41467-026-69244-z

Figure Lengend Snippet: a Effect of naturally occurring small molecule metabolites, chaotropic compounds, and small molecule modulators of protein-protein interactions on phase separation and fibrillization of SynTag-Tau. The chemical structure of the respective small molecule is also shown. b Dose-dependent effect of L-Arg in inhibition of SynTag-Tau condensate-to-amyloid transition. c SynTag-Tau partition coefficient analysis at different L-Arg doses. The thick dashed line of the violin plot represents the median based on data of 53 condensates from three independent replicates, whereas the thinner dotted lines above and below represent the upper and lower quartiles, respectively. d (top) Chemical structure of α-dansyl-L-arginine (dansyl-L-Arg). (bottom) Partitioning of either dansyl chloride (dansyl-Cl) or dansyl-L-Arg doped along with L-Arg in SynTag-Tau condensates, visualized using Alexa594- (A594) labeled SynTag-Tau, at 2 h sample age. e Partition coefficient analysis of either dansyl-Cl or dansyl-L-Arg, either with or without L-Arg in SynTag-Tau condensates. The thick dashed line of the violin plot represents the median based on data of 32 condensates from three independent replicates, whereas the thinner dotted lines above and below represent the upper and lower quartiles, respectively. f L-Arg ethyl ester (L-Arg EE), a derivative of L-Arg that lacks a carboxyl group, failed to prevent condensate aging to fibrils. D-Arginine (D-Arg) treated condensates do not transition to fibrils, similar to the L-Arg condition shown in ( a ). The display range of images shown across these figure panels was adjusted independently for better clarity. Each of these experiments was independently repeated three times. Source data are provided as a Source Data file.

Article Snippet: Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl 2 , and 0.5 mM EGTA, supplemented with 2 mM DTT.

Techniques: Protein-Protein interactions, Inhibition, Labeling

Journal: Nature Communications

Article Title: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

doi: 10.1038/s41467-026-69244-z

Figure Lengend Snippet: a ThT fluorescence measurements at different sample ages, either without (untreated) or with 2 mM L-Arg. Representative images are shown on the left, and corresponding plots of ThT fluorescence intensities are shown on the right. The contrast was adjusted independently across the images to better visualize condensates and fibrils that are very dim. The center line of the box plot represents the median, and the individual data points represent 10 measurements across three independent replicates. The whiskers indicate the full range of the data from the minimum to the maximum. b Individual Z-slices of untreated SynTag-Tau condensates visualized using ThT (green) as well as A594-labeled SynTag-Tau (red). The indicated z-position is relative to the leftmost z-slice in the panel. The inset shown represents the area demarcated by the white-dashed box. Sample age is 96 h. c Maximum intensity projection (MIP) image of 2 mM L-Arg treated SynTag-Tau condensates visualized as a merge of A594-labeled SynTag-Tau and ThT fluorescence. The display range of ThT fluorescence in ( b ) and ( c ) is identical. Sample age is 96 h. d Peak fitting of averaged amide I spectra obtained from BCARS hyperspectral imaging of L-Arg-treated SynTag-Tau condensates reveals changes in protein molecular conformations at 2 h (nascent condensates) and 6 h (aged condensates). Spectra from multiple samples within each independent experiment (n ≥ 6 per experiment) were averaged prior to analysis. Data are presented as mean values with S.E.M representing three independent replicates. e Heat map based on the amide I spectral decomposition obtained from BCARS hyperspectral imaging showing the mean percentages of protein conformations in untreated versus 2 mM L-Arg treated SynTag-Tau condensates at 2 h (nascent) and 6 h (aged) of age. The mean percentages based on measurements from three independent replicates represent the quotient of each conformation’s peak fitted area divided by the cumulative area of β-sheet, random coil, and α-helix. f FD-FLIM map of L-Arg treated condensates at various time points (since sample preparation). g Representative fluorescence lifetime distributions at 1 h and 5 h time points (since sample preparation). The points are the data, and the lines are Gaussian fittings with the corresponding goodness of fit (R 2 ) indicated. Each of these experiments was independently repeated three times. Source data are provided as a Source Data file.

Article Snippet: Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl 2 , and 0.5 mM EGTA, supplemented with 2 mM DTT.

Techniques: Fluorescence, Labeling, Imaging, Sample Prep

Journal: Nature Communications

Article Title: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

doi: 10.1038/s41467-026-69244-z

Figure Lengend Snippet: a MT polymerization assay of SynTag-Tau condensates treated with 1 mM L-Arg at various time points. b A comparison of MT surface coverage as a function of condensate age between untreated SynTag-Tau condensates (Fig. ) and L-Arg treated SynTag-Tau condensates (images corresponding to 2 mM condition are shown in Supplementary Fig. ). Horizontal black lines represent the median value. The individual data points represent measurements based on three independent replicates. Sample size for untreated condition: 1h, 30 datapoints; 2.5h, 24 datapoints; 5h, 23 datapoints; 8 h, 24 datapoints; 12 h, 16 datapoints; 16 h, 14 datapoints. For 1 mM L-Arg condition: 1h, 19 datapoints; 2.5h, 15 datapoints; 5 h, 16 datapoints; 8 h, 15 datapoints; 12 h, 11 datapoints; 16 h, 13 datapoints. For 2 mM L-Arg condition: 1 h, 20 datapoints; 2.5 h, 19 datapoints; 5 h, 15 datapoints; 8 h, 11 datapoints; 12 h, 11 datapoints; 16 h, 15 datapoints. Statistical significance was determined using an unpaired two-sided Student’s t -test between the MT surface coverages of untreated condensates (salmon) and either 1 mM L-Arg treated condensates (green) or 2 mM L-Arg treated condensates (blue) (* means p < 0.05, ** means p < 0.01, ***means p < 0.001, ****means p < 0.0001). The associated P values are shown from left to right: 0.0028, 0.0003, 0.0264, 0.0006, 0.0002, and 0.00009. c Schematic representing the proposed model of biochemical modulation of condensate metastability governing protein condensate-to-fibril conversion and its impact on the condensate biochemical activity in tubulin partitioning (shown as dimers) and MT formation (shown as tubule). These experiments were independently repeated three times. Source data are provided as a Source Data file.

Article Snippet: Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl 2 , and 0.5 mM EGTA, supplemented with 2 mM DTT.

Techniques: Polymerization Assay, Comparison, Activity Assay