Samples of 3 × 108 cells of each culture in asynchronous and arrest conditions were collected and washed with stop buffer (NaCl 150 mM, NaF 50 mM, Ethylene-diamine-tetraacetic acid (EDTA) 10 mM and NaN3 1 mM, pH 8). Pellets were kept at −80°C. The frozen cells were thawed on ice and resuspended in 50 μl of cold lysis buffer (Phosphate buffered saline (PBS) 1X, NaF 50 mM, Ethylene diamine tetraacetic acid (EDTA) 2 mM pH 8, NP-40 1%, p-NH2-benzamidine 1.3 mM, Phenyl methane sulfonyl fluoride (PMSF) 1 mM and a protease inhibitor cocktail (Roche)). Cells were broken using 750 mg of glass beads (0.4 mm, Sigma) for 15 s (three times) in a Fast-prep machine (Bio101 Inc.) and the crude extract was recovered by 0.2 ml of lysis buffer. After 30 min of centrifugation (15 000 rpm), the soluble fraction was collected and kept at −80°C. Protein concentration was determined by Bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce).
Bicinchoninic acid bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, China
The Bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantification method for determining total protein concentration in a sample. The kit utilizes the reduction of Cu2+ to Cu+ by protein in an alkaline medium, and the subsequent colorimetric detection of the cuprous cation (Cu+) using a unique reagent containing bicinchoninic acid.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Glutamine, by Thermo Fisher Scientific (3 mentions)
Tween 20, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Ethyl acetate, by Merck Group (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Bafilomycin a1, by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Coomassie brilliant blue r 250, by Merck Group (3 mentions)
171 protocols using bicinchoninic acid bca protein assay kit
1
Cell Lysis and Protein Extraction Protocol
2
Recombinant Proteins and Peptide Synthesis
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Eimeria tenella elongation factor 1-α (rEF-1α, GenBank Accession Number KX900609) expressed in Escherichia coli (BL21) (Lin et al., 2017 (link)) and recombinant chicken IL-7 (rchIL-7, GenBank Accession number KY02410.1) expressed in Chinese hamster ovary (CHO ) (Panebra et al., 2021 ) (GenScript, Inc., Piscataway, NJ) were used. The concentration of both recombinant proteins (rEF-1α and rchIL-7) was determined using a Thermo-Scientific-Pierce Bicinchoninic Acid (BCA ) protein assay kit (Waltham, MA) at 560 nm. cNK-2 peptide (RRQRSICKQLLKKLRQQLSDALQNNDD) corresponding to the structural domain of chicken NK-lysin with anti-Eimeria was custom synthesized (>96% pure) (Peptide 2.0 Inc., Chantilly, VA) as previously reported (Kim et al., 2017 (link)). The peptide concentration was quantified using a UV Spectro-photometer at 210 nm wavelength.
3
Immunoblotting of Cytochrome P450 Enzymes
4
Radioligand Binding Assay for A2BAR
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Bovine serum albumin (BSA) and the bicinchoninic acid (BCA) protein assay kit were purchased from Fisher Scientific (Hampton, New Hampshire, United States). [3H]PSB-603 (specific activity 79 Ci mmol−1) was custom-labeled and purchased from Quotient Bioresearch (Waltham, MA) and ZM241385 was a kind gift by Zeneca Pharmaceuticals (Macclesfield, United Kingdom). Adenosine deaminase (ADA) was purchased from Sigma Aldrich (Zwijndrecht, the Netherlands). All other chemicals used in the biological experiments were purchased from standard commercial sources.
CHO-spap cells either “empty” or stably expressing the wildtype human A2BAR (CHO-spap-hA2BAR) were kindly provided by S.J. Dowell, Glaxo Smith Kline.
CHO-spap cells either “empty” or stably expressing the wildtype human A2BAR (CHO-spap-hA2BAR) were kindly provided by S.J. Dowell, Glaxo Smith Kline.
5
Radioligand Binding Assay for A2BAR
6
Radiolabeled Nucleoside Uptake Assay
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Bovine serum albumin (BSA) and the bicinchoninic acid (BCA) protein assay kit were purchased from Fisher Scientific (Hampton, New Hampshire, USA). [3H]NBTI (specific activity 33.1 Ci mmol−1) was purchased from PerkinElmer (Groningen, The Netherlands) and NBTI (4-nitrobenzylthioinosine) was obtained from Sigma-Aldrich (Steinheim, Germany). Erythrocytes were obtained from Sanquin (Amsterdam, The Netherlands). All other chemicals were purchased from standard commercial sources.
7
Multicomponent Nanoparticle Formulation
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Baker’s yeast (S. cerevisiae) was provided by Lesaffre Management Co. Ltd. (Shanghai, China). GOx (100 to 250 U/mg) from Aspergillus niger, Cat (2000 to 5000 U/mg) from bovine liver, branched polyethyleneimine (bPEI, molecular weight = 25 kDa), FITC, glucose and CDI, PMA, 5-ASA, Cur, laminarin, and LPS were purchased from Sigma-Aldrich (Missouri, USA). l -α-distearoyl-phosphatidylcholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG2000-NH2) were purchased from Avanti Polar Lipids (Alabama, USA). Sulfo-Cy5.5 N-hydroxysuccinimide (NHS) ester was purchased from Lumiprobe (Maryland, USA). A bicinchoninic acid (BCA) protein assay kit, Amplex Red GOx assay kit, and Amplex Red Cat assay kit were purchased from Invitrogen (Carlsbad, USA). Acrylic polymers (Eudragit L, 100-55) were purchased from Evonik Industries (Hanau, Germany). Caco-2 cells, Raji cells, and THP-1 cells were obtained from the American Type Culture Collection (Manassas, USA). The cell culture reagents were obtained from Gibco (Grand Island, USA). All other chemicals and reagents used were of analytical grade.
8
Epigenetic Regulation of Cell Invasion
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Reagents and materials. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), TRIzol reagent, the TaqMan MicroRNA assay kit, the bicinchoninic acid (BCA) protein assay kit and Lipofectamine ® 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The demethylation drug 5-Aza-2'-deoxycytidine (Aza) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The miRNeasy mini kit was purchased from Qiagen (Valencia, CA, USA). Mouse monoclonal anti-E-cadherin (1:500; cat. no. YM0208), mouse monoclonal Vimentin (1:500; cat. no. YM0645), rabbit polyclonal Caspase 3 (1:500; cat. no. YT0656), mouse monoclonal BH3 interacting-domain death agonist (Bid; 1:500; cat. no. YM0062), rabbit polyclonal phosphorylated B-cell lymphoma-2 (Bcl-2; 1:500; cat. no. YP0031) and mouse monoclonal β-actin (1:500; cat. no. YM3028) antibodies were purchased from ImmunoWay (Cambridge, UK). Goat anti-mouse (1:1,000; cat. no. SA001) and goat anti-rabbit (1:1,000; cat. no. SA009) IgG(H+L)-HRP secondary antibodies were purchased from Auragene Bioscience (Changsha, China). A cell invasion assay kit was purchased from Merck Millipore (Darmstadt, Germany).
9
Streptavidin-Aptamer Biosensor for Malaria Detection
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Streptavidin from Streptomyces avidinii, horseradish peroxidase (HRP), lysozyme, imidazole, Tris powder, 1-fluoro-2,4-dinitrobenzene (FDNB), phenylmethylsulfonyl fluoride (PMSF), sodium bicarbonate, sodium carbonate, sodium phosphate, sodium periodate, and sodium borohydride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ampicillin and isopropyl β-d -1-thiogalactopyranoside (IPTG) were purchased from LPS Solution (Daejeon, Republic of Korea). All enzymes for plasmid construction were purchased from Enzynomics (Daejeon, Republic of Korea). Pyridoxal phosphate and phosphate buffered saline (pH 7.4) were obtained from AMRESCO (Solon, OH, USA), and ethylene glycol was purchased from Junsei Chemical (Tokyo, Japan). Bovine serum albumin (BSA) was purchased from Roche (Basel, Switzerland). The bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was purchased from Kementec (Taastrup, Denmark). The single-stranded DNA aptamer that specifically binds to PfLDH called ‘2008s’ (5′-CTGGGCGGTAGAACCATAGTGACCCAGCCGTCTAC-3′) was synthesized by Integrated DNA Technologies (Singapore, Republic of Singapore), and biotin or HRP was conjugated at 3′-end of aptamer, respectively. Human LDH (hLDH) was purchased from Abcam (Cambridge, UK).
10
Protein Extraction and Quantification
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The cells were washed three times in cold PBS 1X, harvested in cold PBS 1X and centrifuged at 500 x g at 4 °C for 5 min. The pellets were lysed in 8 M urea 10 mM HEPES pH 8.0 and centrifuged at 16000 x g at 4 °C for 10 min. The amounts of protein in the supernatants were measured with Thermo Scientific™ Bicinchoninic acid (BCA) protein assay kit (#23225) and an amount of 50 μg of proteins was kept for each sample. The volumes were adjusted to 100 μL with 100 mM TEAB dissolution buffer (triethyl ammonium bicarbonate #90114).
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