Snap-frozen protein pellets were lysed, reduced, and alkylated in lysis buffer (1% SDC, 10 mM tris(2-carboxyethyl)phosphine hydrochloride), 40 mM chloroacetamide, and 100 mM TRIS, pH 8.0 supplemented with protease inhibitor (cOmplete mini EDTA-free, Roche). Cells were heated at 95 °C and sonicated with a Bioruptor Plus (Diagenode) for 15 cycles of 30 s. Bradford protein assay (Bio-Rad Protein Assay Kit I, Bio-Rad) was used to determine the protein amount, after which samples were split into 10 μg aliquots. Proteins were digested overnight at 37 °C with 1:50 trypsin (Sigma-Aldrich) and 1:75 and lysyl endopeptidase (Wako), after which samples were desalted using an Oasis platform, dried down, and stored at −80 until further use.
Protein assay kit 1
Manufactured by Bio-Rad
Sourced in United States
The Protein Assay Kit I is a colorimetric assay designed for the quantitative determination of protein concentration. It utilizes a copper-based reagent that reacts with protein, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Lysyl endopeptidase, by Fujifilm (2 mentions)
Complete mini edta free, by Roche (2 mentions)
Trypsin, by Merck Group (2 mentions)
Bioruptor plus, by Diagenode (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
Hybond ecl membrane, by Cytiva (1 mentions)
Ecl western blotting detection kit, by Cytiva (1 mentions)
Assaymap bravo platform, by Agilent Technologies (1 mentions)
Phosstop, by Roche (1 mentions)
Deguelin, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Low molecular weight chitosan, by Merck Group (1 mentions)
Protein assay kit 2, by Bio-Rad (1 mentions)
Methylthiazolyldiphenyl tetrazolium bromide mtt, by Merck Group (1 mentions)
Tripolyphosphate tpp, by Merck Group (1 mentions)
20 protocols using protein assay kit 1
1
Protein Extraction and Digestion Protocol
2
Immunoblotting of Tobacco Leaf Proteins
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Total protein was extracted [43] (link) from 21-d-old tobacco leaves. Protein concentration was determined using the Bio-Rad Protein Assay Kit I (Bio-Rad). Protein (20 µg per well) separated on 12% SDS-PAGE was transferred onto Hybond-ECL membrane (Amersham) using a Trans-Blot® cell (Bio-Rad). Antibodies raised against the synthetic peptide (DESYQSRDLEKVSQQ) corresponding to BjHMGS1 amino acids 290 to 304 were used in western blot analyses [4] (link), [44] (link). Cross-reacting bands were detected using the ECL™ Western Blotting Detection Kit (Amersham).
3
Assessing Mitochondrial Enzyme Activities
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The OGDHC activity was assayed at endogenous, i.e., present in cellular lysates, ThDP level, or with 1 mM ThDP added to the assay medium. The former assay estimates intracellular portion of active OGDHC, i.e., the level of the holoenzyme with bound ThDP (holo-OGDHC). The latter assay estimates total amount of both the active (holoenzyme) and latent (apoenzyme) forms of OGDHC. Besides, the total levels of GDH, MDH, NADP+-dependent malic enzyme, NADP+-dependent IDH, NAD+-dependent IDH were measured as described earlier (Bunik et al., 2020a (link)). The enzymatic activities were normalized to total protein content, determined with Bio-Rad Protein Assay Kit I (Bio-Rad, 5000001), and expressed as micromoles of the consumed substrate or generated product per min per mg of protein.
4
Phosphorylated Peptide Enrichment Protocol
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Snap-frozen protein pellets were lysed, reduced, and alkylated in lysis buffer (1% sodium deoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine hydrochloride), 40 mM chloroacetamide, and 100 mM TRIS, pH 8.0 supplemented with phosphatase inhibitor (PhosSTOP, Roche) and protease inhibitor (cOmplete mini EDTA-free, Roche). Cells were heated at 95 °C and sonicated with a Bioruptor Plus (Diagenode) for 15 cycles of 30 s. Bradford protein assay (Bio-Rad Protein Assay Kit I, Bio-Rad) was used to determine the protein amount, after which samples were split into 200 μg aliquots. Proteins were digested overnight at 37 °C with trypsin (1:50 μg/μg) (Sigma-Aldrich) and lysyl endopeptidase (1:75 μg/μg) (Wako). Heavy-labeled phosphopeptides were added to the samples. The SDC was precipitated with 2% formic acid (FA) twice, after which samples were desalted and enriched in an automated fashion using the AssayMap Bravo platform (Agilent Technologies) with corresponding AssayMap C18 (Agilent Technologies) reverse-phase column as previously described (33 (link)). The enrichment of phosphorylated peptides, in short, samples were dissolved in 80% ACN/0.1% TFA. Fe3+ IMAC cartridges were used, using 80% ACN/0.1% TFA as washing buffer and 10% Na4OH as elution buffer.
5
Cytotoxicity and DNA Damage Assays
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3-(4 (link),5 (link))-dimethylthiazol(-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI) and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The total protein extraction and comet assay kits were purchased from Nanjing Keygen Biotech., Co., Ltd. (Nanjing, China). All the chemicals used in this study were analytically pure and of culture grade. The primary antibodies against BRCA1 (monoclonal rabbit anti-human), ERCC1 (polyclonal rabbit anti-human), XRCC1 (polyclonal rabbit anti-human) and GAPDH (monoclonal rabbit anti-human) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), and the Bio-Rad protein assay kit I (cat. no. 500–0001) was purchased from Bio-Rad (Hercules, CA, USA).
Deguelin was purchased from Sigma-Aldrich, dissolved in DMSO as a stock solution and stored at 4°C. The stock solution was then diluted in cell culture medium to a final concentration of 0.05% DMSO (V/V).
Deguelin was purchased from Sigma-Aldrich, dissolved in DMSO as a stock solution and stored at 4°C. The stock solution was then diluted in cell culture medium to a final concentration of 0.05% DMSO (V/V).
6
Membrane Protein Extraction Protocol
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To prepare membrane proteins, 300 mL cultures were harvested by centrifugation (7200 × g for 10 min at 4°C). The cells were washed three times with cold Tris–HCl (pH 9.5) plus protease inhibitor cocktail (Roche, Germany), resuspended in cold Tris–HCl (pH 9.5) containing protease inhibitor cocktail and sonicated on ice under the following conditions: 2 s of sonication with a 2 s interval set at 30% duty cycle. Every cycle lasts for 2 min with a 2 min interval. After the cells were completely split, the cell lysate was centrifuged at 15 000 × g for 20 min at room temperature to collect the insoluble pellet. The pellet was solubilized with lysis buffer (5 M urea, 2 M thiourea, 2% w/v CHAPS, 2% w/v SB 3–10, 40 mM Tris, and 2 mM TBP) after washing twice with cold Tris–HCl (pH 9.5). The mixture was vortexed for 5 min and centrifuged at 15 000 × g for 10 min at room temperature to pelletize the insoluble components. The supernatant was recovered, and the protein concentration was assayed with a Bio-Rad Protein Assay Kit I (Biorad, USA). Three separate biological replicates were performed for 2-DE analysis.
7
Chitosan-TPP Nanoparticle Antibacterial Assay
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Low-molecular-weight chitosan (LMW, 50–190 kDa; degree of deacetylation, DD = 75–85%), tripolyphosphate (TPP), and acetic acid were purchased from Sigma-Aldrich (St Louis, USA). Gonococcus medium (GC) agar, supplemented with 1% IsoVitaleX and gonococcus broth medium (GCP) was purchased from (Difco). All of the other solvents and chemicals were of analytical grade. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic–antimycotic solution was purchased from Gibco (NY, USA). Synthetic water-soluble peptides C7-3 (H-ACNYCRLNLWGGGS-NH2), C7-3m1 (H-ACNYSRLNLWGGGS-NH2), and C7-3m2 (H-ACSY CRLNLWGGGS-NH2) were purchased from Pepmic Co., Ltd (Suzhou, China). A control strain of N. gonorrhoeae, NCTC 12,700, and the NCTC 14,208 N. gonorrhoeae reference strain were purchased from Public Health England (PHE). Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad Protein Assay Kit I, and Bio-Rad Protein Assay Kit II were purchased from Bio-Rad (USA). HeLa (human cervical cancer epithelial cells) was purchased from the American Type Culture Collection (ATCC CCL-2). Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) was purchased from Sigma-Aldrich (Saint Louis, USA).
8
Fractionation of Cellular Proteins
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Fractionation of cytoplasmatic and nuclear proteins was performed using three buffers: HB-Buffer (10 mM Tris-HCl, pH 8, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM 2-mercaptoethanol, protease inhibitor [P8340, Merck; 4 µL per 5 mL]); Lysis-Buffer: HB-Buffer + 0.4% NP-40); Buffer C (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitor [P8340, Merck; 4 µL per 1 mL]). 1–5 × 106 cells were harvested, pelleted at 200× g for 5 min and washed at RT. The cell pellet was resuspended in 350 µL HB-Buffer and centrifuged at 200× g for 1 min at 4 °C. The supernatant was removed and the pellet was resuspended in 100 µL Lysis-Buffer followed by incubation for 15 min at 4 °C. The supernatant containing the cytosolic fraction was harvested by centrifugation at 15,000× g for 5 min at 4 °C. The pellet was washed twice with 75 µL Lysis-Buffer, resuspended in 60 µL Buffer C and incubated for 15 min at 4 °C. Following centrifugation at 15,000× g for 5 min at 4 °C the supernatant containing the nuclear fraction was harvested. Protein concentrations were then determined using the Protein Assay Kit I (Bio Rad, 5000001).
9
PrPC Deglycosylation and Western Blot
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Cells were lysed with homogenizing buffer (Tris HCl 50 uM, NaCl 150 mM, EDTA 1 mM, DOC 0.25%, NP40 1%) supplemented with cOmplete™ protease inhibitor cocktail (Roche, Basel, Switzerland). The protein concentration was determined with the Protein Assay kit I (Bio-Rad, Hercules, CA, USA). For each sample, 20 µg protein was deglycosylated with PNGase-F (New England BioLabs Inc) in accordance with the manufacturer’s guidelines, and 20 µg protein were analysed untreated. The samples were denatured using sodium dodecyl sulphate (SDS) loading buffer (Invitrogen) and Sample Reducing Agent (Thermo Fisher Scientific) before separation by polyacrylamide gel electrophoresis (PAGE) on a 12% Criterion™ XT Bis-Tris gel (Bio-Rad). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Chicago, IL, USA) and the membrane blocked with 5% non-fat dried milk in TBS – Tween. The membrane was then incubated with primary antibody (P4 mouse anti PrPC; Ridascreen Biopharm AG, Darmstadt, Germany) 1:1000, and secondary antibody (anti-mouse IgG conjugated to alkaline phosphatase; Invitrogen) 1:1000. Detection with EFC™ substrate (GE Healthcare) was performed on a Typhoon 9200 imager (Amersham Biosciences, Sunnyvale, CA, USA).
10
Protein Quantification using Bio-Rad Assay
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The total protein content was determined using the Bio-Rad Protein Assay Kit I, 5000001, according to manufacturer’s instructions.
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