Human iPSCs (201B7) cultured for 7 days were lysed in RIPA buffer (20 mM Tris/HCl (pH 7.6) (35,436-01, Nacalai Tesque), 150 mM NaCl (31,320-05, Nacalai Tesque), 0.1 % SDS (31,606-75, Nacalai Tesque), 1 % NP-40 (25,223-75, Nacalai Tesque), 1 mM MgCl2 (20,909, Nacalai Tesque), and protease inhibitor (25,955-11, Nacalai Tesque)). Cell lysates were mixed with amylose resins conjugated with recombinant MBP-tagged bFGF-WT protein and incubated at 4 °C for 1 h. After washing the resins, bound proteins were eluted with SDS sample buffer (125 mM Tris-base (35,434-21, Nacalai Tesque), 960 mM glycine (17,109-35, Nacalai Tesque), and 17.3 mM SDS (31,606-75, Nacalai Tesque)) for immunoblotting.
Sodium dodecyl sulfate (sds)
Manufactured by Nacalai Tesque
Sourced in Japan
SDS is a laboratory equipment used for the separation and analysis of proteins. It functions by denaturing proteins and separating them based on their molecular weight through gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Dithiothreitol (dtt), by Fujifilm (3 mentions)
Sodium deoxycholate, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Congo red, by Nacalai Tesque (2 mentions)
Np 40, by Nacalai Tesque (2 mentions)
Sodium chloride (nacl), by Nacalai Tesque (2 mentions)
Nonidet p 40, by Nacalai Tesque (2 mentions)
Glycerol, by Nacalai Tesque (2 mentions)
Tris hydroxymethyl aminomethane, by Nacalai Tesque (2 mentions)
Sodium pyrophosphate, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Sodium fluoride, by Fujifilm (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Fujifilm (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
19 protocols using sodium dodecyl sulfate (sds)
1
Affinity Purification of bFGF-Binding Proteins
2
Microvessel Protein Extraction and Quantification
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The microvessels were homogenized in phosphoprotein lysis buffer containing 10 mM Tris-HCl (pH 6.8; Nacalai Tesque; 35434–34), 100 mM NaCl (Sigma; 28-2270-5), 1 mM EDTA (pH 8.0; Wako; 311–90075), 1 mM EGTA (Wako; 346–01312), 10% glycerol, 1% Triton-X100 (Sigma; X100), 0.1% sodium dodecyl sulfate (SDS; Nacalai Tesque; 02873–75), 0.5% sodium deoxycholate (Sigma; D6750), 20 mM sodium pyrophosphate (Sigma; S6422), 2 mM sodium orthovanadate (Sigma; S6508), 1 mM sodium fluoride (Wako; 196–01975), 1% protease inhibitor cocktail (Sigma; P2714), 1% phosphatase inhibitor cocktail 2 (Sigma; P5726), 1% Phosphatase Inhibitor Cocktail 3 (Sigma; P0044), and 1 mM PMSF (Sigma) using an electric mixer, and then sonicated on ice. Samples were centrifuged at 15,000 × g for 15 min at 4°C, and the supernatants were collected. The total protein concentration in the lysates obtained from microvessels was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific; 23225).
3
Cell Proliferation Evaluation by DNA Assay
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The number of cells proliferated in GHNF and PPNF was evaluated by the DNA assay [38 (link),39 (link)]. Briefly, the cell-GHNF and PPNF constructs cultured were washed with the PBS solution three times, and stored at −30 °C until to the assay. After thawing, cells in the GHNF and PPNF were lysed by 30 mM saline-sodium citrate (SSC) containing 0.2 mg/ml sodium dodecyl sulfate (Nakalai Tesque Inc., Japan) at 37 °C, 300 rpm for 24 h by thermomixer (Eppendorf Co. Ltd., Germany). The dye solution of 30 mM SSC containing 1.25 μl/ml Hoechst 33,258 DMSO solution (1 mg/ml, Nakalai tesque, Japan) (160 μl) and the cell lysate (40 μl) was mixed in each well of 96 multi well black flat bottom plate (Corning Inc., NY), while the fluorescence intensity of mixed solution was measured at excitation and emission wavelengths of 355 and 460 nm on fluorescence spectrometer (SpectraMax; Molecular Devices, California, USA). The number of cells was calculated by a standard curve between the fluorescent intensity and cell number known. The DNA assay was performed for 4 materials at each time point and the average number of cells was calculated.
4
Rubber-based Graphene Nanocomposites
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The natural rubber used in this work is high-ammonia natural rubber latex (HANR, dry rubber content (DRC) 60%) supplied by Dau-Tieng rubber company. Sodium dodecyl sulfate (SDS, 97%) was bought from Nacalai Tesque, Japan. Graphite flake powder (>99% purity) was obtained from Yen-Bai province, Vietnam. The compounds KMnO4, NaNO3 (analytical grade), and TEPA were purchased from Sigma-Aldrich. Vinyltriethoxysilanes, tert-butyl hydroperoxide (TBHPO), and urea were purchased from Tokyo Chemical Industry, Japan.
5
Tissue-Hydrogel Hybridization Protocol
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Brain slices were incubated at 4°C overnight in A4P0 hydrogel (4% [w/v] acrylamide [161-0140, Bio-Rad] and 0.25% 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride [VA-044, Wako Pure Chemical Industries] in PBS(−)). The slices were vacuum degassed for 10 min, placed under nitrogen for 10 min, and incubated at 37°C for 3 hr to initiate tissue-hydrogel hybridization. After washing twice with PBS(−) for 15 min at 20–25ºC, the slices were incubated at 37°C for 24 hr in 8% (w/v) sodium dodecyl sulfate (31607-65, Nacalai Tesque) in PBS(−) (Yang et al., 2014 (link)).
6
Compound Preparation for Cell Assays
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Primaquine and erythromycin were obtained from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Aflatoxin B1 and dimethyl fumarate were obtained from Sigma–Aldrich (St. Louis, MO, USA). Terbinafine was obtained from Tokyo Chemical Industry (Tokyo, Japan). These compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma–Aldrich). The final concentration of DMSO in culture medium was less than 0.1%. SDS and D-mannitol were purchased from Nacalai Tesque (Kyoto, Japan) and dissolved in distilled water.
7
Yeast Cell Protein Extraction
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After yeast was cultured in syringes, the appropriate number of cells was collected and they were suspended in 100 µL of 0.2 m NaOH (Wako) and 1% 2‐mercaptoethanol (Wako) per OD, placed on ice for 10 min, and centrifuged at 17 800 g at 4 °C for 2 min. The supernatant was discarded, and 100 µL of 1× sample buffer (2% SDS (Nacalai Tesque), 100 mm DTT (Wako), 60 mm Tris/HCl (pH 6.8) (Sigma), 0.001% bromophenol blue (Sigma), 10% glycerol (Wako)) was added per 1 OD. The pellet was suspended by adding 100 µL per OD, heated at 100 °C for 5 min, and the centrifuged supernatant was used as the sample for SDS/PAGE.
8
Immunoprecipitation of c-Myc Protein
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The procedure was performed as described previously (Shimmoto et al, 2009 (link)). For immunoprecipitation, ∼1.0 × 108 cells from 50 ml culture were harvested and washed once with PBS. The cells were then re-suspended in 0.5 ml of IP buffer (20 mM HEPES-KOH [pH 7.6] [Nacalai tesque], 50 mM potassium acetate [Sigma-Aldrich], 5 mM magnesium acetate [FUJIFILM Wako], 0.1 M sorbitol [FUJIFILM Wako], 0.1% TritonX-100 [Sigma-Aldrich], 2 mM DTT [FUJIFILM Wako], 20 mM Na3VO4 [Sigma-Aldrich], 50 mM β-glycerophosphate [Sigma-Aldrich], and Protease Inhibitor Cocktail [Sigma-Aldrich]) and were disrupted with glass beads using a multi-beads shocker (Yasui Kikai). The lysates were cleared by centrifugation (20,000g for 10 min at 4°C). The supernatants of lysates were mixed with anti-c-Myc antibody (Nacalai tesque) attached to Protein G Dynabeads (10004D; Thermo Fisher Scientific). After incubating for 1 h, the beads were washed with IP buffer and proteins were extracted by boiling with 1× sample buffer (2% SDS [Nacalai Tesque], 4 M Urea [Nacalai Tesque], 60 mM Tris–HCl [pH 6.8] [Nacalai Tesque], 10% Glycerol [Nacalai Tesque], and 70 mM 2-mercaptethanol [Sigma-Aldrich]).
9
Conidial Stress Tolerance Assay
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A conidial suspension of the control or ΔAohok1 strain (~103 or 105/10 µl) was spotted onto each medium plate and incubated at 20 °C, 30 °C or 37 °C for 3 to 8 days. To test cell wall stress tolerance, either 300 µg/ml of Calcofluor White (Sigma), 90 µg/ml of SDS (Nacalai tesque) or 90 µg/ml of Congo Red (Nacalai tesque) was added to M agar plates.
10
Western Blot Analysis of BRCA2 and FANCM
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Samples were prepared in RIPA buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA (pH 8.0), 1% NP-40 (Nacalai Tesque), 1% SDS (Nacalai Tesque), 1% sodium deoxycholate (Sigma–Aldrich), supplemented with cOmplete, Mini (Roche)) or RIP Lysis buffer (20 mM Tris-HCl (pH 7.4), 100 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, supplemented with cOmplete, Mini (Roche)). Samples were mixed with loading buffer containing β-mercaptoethanol, boiled at 95 °C for 5 min, and cooled on ice. Samples were loaded on 5–20% polyacrylamide gels (ATTO), electrophoresed, and transferred to PVDF membranes (Bio-Rad). For BRCA2 and FANCM proteins, samples were loaded on NuPAGE 3–8% Tris-Acetate Protein Gels (Thermo Fisher Scientific). The membranes were immersed in 5% skim milk (BD Difco) in TBS-T. Signal Enhancer HIKARI for Western Blotting and ELISA (Nacalai Tesque) or 5% skim milk in TBS-T was used to dilute primary and secondary antibodies. Blots were developed using Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer’s instructions. Chemiluminescent detection was performed using Amersham Imager 600 (GE Healthcare).
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