PAFA was conducted according to the method described elsewhere39 (link). Ten µg mouse recPrP was denatured to unfolded using 6 M guanidine hydrochloride (GdnHCl) (Sigma-Aldrich) for 5 min at room temperature and then mixed in 200 µl reaction buffer [4M GdnHCl (Sigma-Aldrich) and 10 µM ThT (Sigma-Aldrich) in phosphate buffered saline (PBS, pH7.4) (Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea)] together with compounds (1–100 µM) and vehicle, DMSO. The PAFA reaction mixture was prepared in a 96-well black flat bottom polystyrene not treated microplate (Corning, Corning, NY, USA), sealed with a microplate adhesive film (USA Scientific, Ocala, FL, USA) and incubated at 37 °C for 60 h with shaking at 335 rpm in Infinite M200 Pro Fluorescence reader (Tecan, Maennedorf, Zurich, Switzerland). The fibrils formation was measured every 1 h by top reading of the fluorescence intensity using a 444 nm excitation and 485 nm emission filter.
Gdnhcl
Manufactured by Merck Group
Sourced in United States
GdnHCl is a lab equipment product manufactured by Merck Group. It is a chemical compound commonly used in various research and analytical applications. The core function of GdnHCl is to serve as a denaturant, which is a substance that can disrupt the non-covalent interactions within proteins, leading to the unfolding of their native structure.
Automatically generated - may contain errors
Lab products found in correlation
E8875, by Merck Group (2 mentions)
C7698, by Merck Group (2 mentions)
Antimycin a, by Merck Group (2 mentions)
M2p labs, by BMG Labtech (2 mentions)
96 well plate, by Greiner (2 mentions)
Gdnhcl, by GraphPad (2 mentions)
Imidazole, by Merck Group (1 mentions)
Adenosine 5 triphosphate atp, by Merck Group (1 mentions)
Bis ans, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Microplate adhesive film, by USA Scientific (1 mentions)
Infinite m200 pro fluorescence reader, by Tecan (1 mentions)
Dextran 70, by Merck Group (1 mentions)
Dextran 40, by Merck Group (1 mentions)
Peg 8000, by Merck Group (1 mentions)
Bolt lds sample buffer and dtt, by Thermo Fisher Scientific (1 mentions)
Sarkosyl 20, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
13 protocols using gdnhcl
1
Protein Aggregation Fluorescence Assay
2
Protein Denaturation with GdnHCl
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3
Yeast Growth and Heat Shock Conditions
4
Protein Aggregation Assay with GdnHCl
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The 10% brain homogenate was centrifuged at 700 g for 5 min, and the supernatant was collected. Equal amounts of brain homogenate were added to increasing concentrations of guanidine hydrochloride (GdnHCl) (Sigma), and the samples were shaken at 800 rpm for 2 h at room temperature. The concentration of GdnHCl was diluted to 0.4 M using RIPA buffer, and the total volume of each sample was made equal by adding 0.4 M GdnHCl. Samples were treated with 50 μg/ml PK for 1 h at 37°C, and the reaction was stopped using PMSF (1 mM final concentration). The samples were centrifuged at 100,000 g at 4°C, the pellet was resuspended in PBS, and 6× SDS loading buffer was added.
5
Unfolding of Photoreceptor Proteins
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The protein unfolding was initiated by mixing 50 µL of the native protein with 500 µL of a buffer solution containing the desired concentration of GdnHCl (Sigma-Aldrich, St. Louis, MO, USA). The concentration of the stock GdnHCl solution was determined by the refraction coefficient. The dependences of the chromophore absorbance, fluorescence and ellipticity at 222 nm on the GdnHCl concentration for the BphP1-FP and its variants were recorded at 23 °C after protein incubation in a solution of an appropriate denaturant concentration at 23 °С for 24 h. Further increases in the equilibration time did not result in noticeable changes in the detected characteristics. The recorded fluorescence intensity was corrected for the primary inner filter effect according to the approach in [43 (link),44 (link)].
6
Protein purification from E. coli
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Rosetta [λDE3] E. coli strain, pET21a and pET21d plasmids were purchased from Novagen (WI, USA). Ampicillin and chloramphenicol were obtained from USB (OH, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG), lysozyme, DNAse, p-methyl-sulfonylfluoride (PMSF), imidazole, Gdn.HCl, bis-ANS and adenosine 5′-triphosphate (ATP) were purchased from Sigma (MO, USA). The unlabeled and 5′-carboxyfluorescein (5-FAM)-labeled ssDNA oligo(dT)20 were purchased from IDT (IA, USA).
7
Purification and Characterization of iRFP713
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iRFP713 and its variants in the holo- and apoforms were expressed and purified as described previously (Stepanenko et al., 2014 (link)) (Supplemental Methods 1 ). SDS/PAGE in a 12% polyacrylamide gels was used to confirm that the purity of the target proteins was at least 95% (Laemmli, 1970 (link)). The concentrated protein was stored in 20 mM Tris/HCl buffer, 150 mM NaCl, pH 8.0. The measurements were carried out at a low protein concentration (absorbance was kept less than 0.1, which corresponds to a protein concentration of 0.14 mg/ml) in 20 mM Tris/HCl buffer, pH 8.0, 1 mM tris(2-carboxyethyl)phosphine (TCEP).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).
8
Yeast Growth and Heat Shock Conditions
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Yeast strains and plasmids used in this work were obtained using standard molecular biology protocols and are listed in Supplementary Tables S1 and S2 , respectively. Unless otherwise stated, yeast cells were grown at 30 °C in synthetic defined (SD) media with 2% glucose (2% glucose, 0.5% NH4-sulfate, 0.17% yeast nitrogen base, and amino acids), as previously described [4 (link)]. Where indicated, 2% glucose in SD media was substituted with 2% ethanol. When grown exponentially, Cdc19irrev strains were never allowed to grow above an optical density 600 (OD600) of 0.8 to avoid stress conditions. Heat shock was induced by shifting exponentially growing cells (OD600 0.4-0.6) for 30 min to 42 °C. Where indicated, cycloheximide (Sigma-Aldrich, C-7698) or Antimycin A (Sigma-Aldrich A8674) was added to the medium at 25 μg/ml or 2 μM final concentration (1 μM in SD plates), respectively. Hsp104 activity was inhibited with 5 mM Gdn-HCl (Sigma-Aldrich 50950) for 3 h prior to stress initiation. Growth rates were determined at 30 °C by measuring OD600 in a 96-well plate (Greiner Bio-One) using a plate reader (BioLector m2p-labs or ClarioStar BMG Labtech), or by imaging serial dilution spottings onto appropriate SD plates after 3 days. Protein overexpression from estradiol-inducible promoters was achieved by exposing cells to 10 mM estradiol (Sigma-Aldrich E8875) for 3 hours.
9
Guanidine-Based Prion Protein Extraction
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50 μL of (1) FFI-BH (10-fold concentrated by means of high-speed centrifugation), (2) FFI-BH_PMCA, and (3) brain homogenates of BvPrP-Tg407-inoculated mice were treated with 450 μL of guanidine hydrochloride (Gdn-HCl; Sigma) at different molar concentrations (1.5, 3, 4.5, and 6 M) for 2 hr at 25°C under shaking (550 rpm). Subsequently, an equal volume of sarkosyl 20% (Sigma) was added to the preparation that was incubated for 10 min with gentle shaking. Samples were centrifuged at 100,000 × g for 1 hr at 4°C. Pellets were washed with PBS and then centrifuged at high speed (100,000 × g, 30 min at 4°C). The resulting pellets were suspended in 50 μL of loading buffer (Bolt LDS Sample Buffer and DTT, ThermoScientific) and then subjected to Wb analysis as described. Membranes were immunoblotted using the 6D11 antibody. Each densitometric analysis of the resulting bands was performed (at least three times per sample) using ImageJ software.
10
Probing PmPV1 Unfolding Dynamics
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The intrinsic fluorescence emission of PmPV1 tryphtophans was used to follow the PmPV1 denaturation induced by guanidine hydrochloride (GdnHCl) (Sigma). Chemical denaturation was performed by incubating overnight 50 µg/mL PmPV1 in the presence of increasing concentrations (0–6 M) of GdnHCl buffered with 0.1 M phosphate buffer at pH 7.4 at 8 °C.
Protein intrinsic fluorescence spectra were recorded on an Olis-upgraded SLM4800 spectrofluorometer (Olis Inc., Bogart, GA) coupled with a Lauda Alpha RA 8 thermostatic bath. Fluorescence spectra were recorded in emission scanning mode at 25 °C. Tryptophan emission was excited at 295 nm (8 nm slit) and recorded between 315 and 436 nm (8 nm slit). Three spectra were recorded and averaged for each sample. The corresponding buffer blank was subtracted. At least two independent samples were measured.
Spectra were characterized by their center of mass (CM) calculated using equation (1 ). Where “I” is fluorescence intensity and “λ” represents the wavelength.
The populations associated with the unfolded fraction (ƒu) were calculated from the CM using equation (2 ). Where “x” represents GdnHCl molarity in each condition.
Protein intrinsic fluorescence spectra were recorded on an Olis-upgraded SLM4800 spectrofluorometer (Olis Inc., Bogart, GA) coupled with a Lauda Alpha RA 8 thermostatic bath. Fluorescence spectra were recorded in emission scanning mode at 25 °C. Tryptophan emission was excited at 295 nm (8 nm slit) and recorded between 315 and 436 nm (8 nm slit). Three spectra were recorded and averaged for each sample. The corresponding buffer blank was subtracted. At least two independent samples were measured.
Spectra were characterized by their center of mass (CM) calculated using equation (
The populations associated with the unfolded fraction (ƒu) were calculated from the CM using equation (
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