To evaluate MMP-2 and 9 total gelatinase activity, a zymography was performed using the methodology described in [62 (link)] with modifications as depicted in Figure 2 . CM concentrated samples were resolved by electrophoresis under non-reducing conditions, in a 10% Polyacrylamide gel containing 0.1% SDS and 0.1% (w/v) gelatin. After electrophoresis, the lanes were individualized and washed with 2.5% Triton X-100 in 3 steps of 20 min, to remove SDS. Gel lanes were incubated for 24 h at 37 °C in Developing Buffer (50 mM Tris Base, 200 mM NaCl, 5 μM ZnCl2, 5 mM CaCl2.2H2O and 0.02% NaN3) containing [15]pyN5, [16]pyN5 and ARP-100 (5–20 µM). Incubation with developing buffer restores MMPs’ protease activity. Therefore, incubations in the absence of compounds were also performed in parallel as control. Finally, gels were stained with 0.1% Coomassie Brilliant Blue R-250 (National Diagnostics, Charlotte, NC, USA). MMP activity was detected as a clear band in the background of uniform staining. Band quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Coomassie brilliant blue r 250
Manufactured by National Diagnostics
Sourced in United States
Coomassie Brilliant Blue R-250 is a protein-binding dye used for the detection and quantification of proteins in various biochemical applications, such as gel electrophoresis and Western blotting. It is a bright blue dye that binds non-specifically to proteins, allowing for the visualization and analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Perfection v700 scanner, by Epson (5 mentions)
42.2ti rotor, by Beckman Coulter (4 mentions)
Immobilon p, by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Sigmaplot 13, by Grafiti LLC (1 mentions)
Tl 100 ultracentrifuge, by Beckman Coulter (1 mentions)
V700 scanner, by Epson (1 mentions)
Microfuge, by Beckman Coulter (1 mentions)
Tla100 rotor, by Beckman Coulter (1 mentions)
Perfection v700 photo scanner, by Epson (1 mentions)
Superose 6 increase 10 300 gl gel filtration column, by GE Healthcare (1 mentions)
Bl21 codonplus de3 ripl competent cells, by Agilent Technologies (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
11 protocols using coomassie brilliant blue r 250
1
Gelatin Zymography for MMP-2 and MMP-9 Analysis
2
Western Blot Analysis of C. elegans Tropomyosin
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Fifteen adult worms were suspended in 15 μl of SDS lysis buffer (2% SDS, 80 mM Tris-HCl, 5% β-mercaptoethanol, 15% glycerol, 0.05% bromophenol blue, pH 6.8), heated at 97°C for 2 min, homogenized briefly by sonication, and subjected to SDS–PAGE (12% acrylamide gel). The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The membrane was blocked in 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and incubated for 1 h with anti-C. elegans tropomyosin (LEV-11) guinea pig polyclonal antibody (Ono and Ono, 2002 (link)). After being washed with PBS-T, the membrane was treated with horseradish peroxidase–conjugated goat anti-guinea pig IgG (MP Biomedicals) for 1 h. The reactivity was detected with Chemi-Lumi One Ultra (Nacalai USA) and exposure to x-ray films. Finally, the membrane was stained with 0.1% Coomassie Brilliant Blue R-250 (National Diagnostics) in 50% methanol and washed in a destaining solution containing 10% acetic acid and 50% ethanol to visualize total proteins (Welinder and Ekblad, 2011 (link)).
3
Actin-binding Protein Interaction Assay
4
Quantitative Western Blot Analysis of Actin
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Ten adult worms were suspended in 15 µl SDS lysis buffer (2% SDS, 80 mM Tris-HCl, 5% β-mercaptoethanol, 15% glycerol, 0.05% bromophenol blue, pH 6.8), heated at 97°C for 2 min, homogenized briefly by sonication, heated again at 97°C for 2 min, and subjected to SDS-PAGE (12% acrylamide gel). The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was blocked in 5% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) and incubated for 1 hr with anti-actin mouse monoclonal antibody (C4, MB Biomedicals, catalog # 08691001; RRID:AB_2335127) at a 1:3000 dilution. The membrane was washed with PBS-T, treated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 dilution) (Pierce/Thermo Scientific, catalog #31430) for 1 hr, and washed with PBS-T. The reactivity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and exposure to X-ray films. Finally, the membrane was stained with 0.1% Coomassie Brilliant Blue R-250 (National Diagnostics) in 50% methanol and destained in a solution containing 10% acetic acid and 50% methanol to visualize total proteins (
Welinder & Ekblad, 2011 (link)). The blots were scanned by an Epson Perfection V700 scanner at 300 dpi., and band intensity was quantified using
ImageJ 1.47v.
Welinder & Ekblad, 2011 (link)). The blots were scanned by an Epson Perfection V700 scanner at 300 dpi., and band intensity was quantified using
5
Actin-Binding of LEV-11 Proteins
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Various concentrations of LEV-11 proteins were incubated with or without 5 μM F-actin in F-buffer for 1 h at room temperature and ultracentrifuged at 42,000 rpm for 20 min using a Beckman 42.2Ti rotor. Supernatants and pellets were separated, adjusted to the same volumes, and examined by SDS–PAGE (12% acrylamide gel). Protein markers (#29458-24; Nacalai USA) were used as molecular mass markers. The gels were stained with Coomassie Brilliant Blue R-250 (National Diagnostics) and scanned by an Epson Perfection V700 scanner at 300 dpi. Because LEV-11 proteins migrated too close to actin in the pellet fractions (see Figure 3B ), actin-dependent depletion of LEV-11 proteins was densitometrically quantified by ImageJ from the supernatant fractions to estimate portions of LEV-11 proteins in the pellets. Actin-independent sedimentation of LEV-11 proteins was quantified from the experiments without actin and subtracted from the data. The data were fitted to the following Hill equation (three-parameter sigmoidal) using SigmaPlot 13 (Systat Software):
where v = bound LEV-11 relative to actin (μM/μM), n = maximal bound LEV-11 relative to actin (μM/ μM), nH = Hill coefficient, KD = dissociation constant, and [LEV-11] = [LEV-11]free.
where v = bound LEV-11 relative to actin (μM/μM), n = maximal bound LEV-11 relative to actin (μM/ μM), nH = Hill coefficient, KD = dissociation constant, and [LEV-11] = [LEV-11]free.
6
Actin Bundling and Binding Assay
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F-actin (10 μM) was incubated with various concentrations of UNC-87A or UNC-87B in F-buffer (0.1 M KCl, 2 mM MgCl2, 1 mM dithiothreitol [DTT], 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–KOH, pH 7.5) for 30 min at room temperature. The reactions were centrifuged either at low speed (18,000 rpm for 10 min using a Beckman Microfuge) to examine actin bundling or at high speed (80,000 rpm for 20 min using a Beckman TL-100 ultracentrifuge with a TLA-100 rotor) to examine F-actin binding. The supernatants and pellets were adjusted to the same volumes and analyzed by SDS–PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (National Diagnostics, Atlanta, GA) and scanned by an Epson V700 scanner at 300 dots/inch, and the band intensity was quantified by ImageJ (National Institutes of Health, Bethesda, MD).
7
Protein Purification via Gel Filtration
8
Fpn Sumoylation Domain Expression
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To express the Fpn sumoylation domain, pGex-5x-FpnSumoD was transformed into BL21-CodonPlus (DE3)-RIPL competent cells (Agilent). GST-fusion protein was induced with 0.1 mM IPTG for 3 h; as control GST was similarly expressed from the vector backbone. Cells were lysed in PBS/0.5% Triton-X100/100 μg per ml lysozyme and total lysates of GST or GST-FpnSumoD were used for pull-down assays using 500 μg/ml recombinant SUMO1 or SUMO2 immobilized on agarose. The agarose beads were washed 3x with GST Bind/Wash buffer (Novagen, UK) by spinning at 13,000 rpm for 1 min each, and bound protein complexes were eluted by resuspending the matrix in 2x NUPAGE loading buffer (Invitrogen) and heated for 10 min at 70 °C. Proteins were resolved on 4–12% NuPAGE Tris-MES gels (Invitrogen) and analyzed by staining with Coomassie Brilliant Blue R-250 (National Diagnostics, Nottingham, UK).
9
F-Actin Binding Analysis of MBP and CAS-2 Variants
10
Actin-Binding Protein Sedimentation Assay
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Filamentous actin (5 or 10 M) was incubated with proteins of interest in F-buffer (0.1 M KCl, 2 mM MgCl2, 20 mM HEPES-KOH, pH 7.5, 1 mM DTT) at room temperature. To examine F-actin binding of the proteins of interest by high-speed co-sedimentation, the samples were ultracentrifuged at 42,000 rpm (200,000 x g) for 20 min using a Beckman 42.2Ti rotor. To examine F-actin bundling by low-speed sedimentation, the samples were centrifuged at 15,000 rpm (18,000 x g) for 10 min using a Hettich MIKRO 200 centrifuge. Supernatants and pellets were separated, adjusted to the same volumes, and examined by SDS-PAGE (12% acrylamide gel). For quantitative analysis of high-speed F-actin cosedimentation, control experiments were performed without actin, and non-specific sedimentation of CLIK-1 was determined and subtracted from sedimentation of CLIK-1 in the presence of actin. The gels were stained with Coomassie Brilliant Blue R-250 (National Diagnostics) and scanned by an Epson Perfection V700 scanner at 300 dpi. Band intensity was quantified with ImageJ.
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