E. coli BL21 (DE3) cells carrying the plasmids of interest were grown in LB at 37 °C to an OD600 of 0.6. Then, IPTG was added at a final concentration of 1 mM to induce protein expression, the temperature was decreased to 26 °C and the culture was incubated for 6 h. Samples (10 mL) were taken at 0 and 6 h of induction and centrifuged. The pellets were resuspended in 1 mL of Tris-HCl buffer (10 mM, pH 7.8) supplemented with protease inhibitor (NZYTech) and lysed by sonication on ice (35% amplitude, 3 s ON plus 9 s OFF for a total of 5 min ON) using a microtip probe linked to a Vibra-cell processor (Sonics). After sonication, samples were centrifuged and resuspended, and the protein of the soluble and insoluble fractions was quantified using the Bradford reagent (Panreac). The expression levels were evaluated through SDS-PAGE (4% stacking gel and 10% running gel). Soluble and insoluble protein fractions were mixed with 2 x sample buffer (65.8 mM Tris–HCl pH 6.8, 26.3% glycerol, 0.01% bromophenol blue, 2.1% SDS, 5% β-mercaptoethanol) and denaturated at 95 °C in the heating block for 5 min. The protein ladders used were NZYColour Protein Marker II (NZYTech) and Blue Prestained Protein Standard—Broad Range (NEB). The gel was stained with Coomassie Blue R-250 for 15 min and de-stained using distilled water.
Nzycolour protein marker 2
Manufactured by NZYTech
Sourced in Portugal
NZYColour Protein Marker II is a pre-stained protein molecular weight marker. It is designed for use in SDS-PAGE and Western blotting applications to estimate the molecular weights of unknown protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by ITW Reagents (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Vibra cell processor, by Sonics (1 mentions)
Bradford reagent, by ITW Reagents (1 mentions)
Porcine trypsin, by Promega (1 mentions)
Nuclease mix, by GE Healthcare (1 mentions)
Destreak reagent, by GE Healthcare (1 mentions)
Bluesafe, by NZYTech (1 mentions)
Ipg buffer ph 4 7, by GE Healthcare (1 mentions)
Phosstop, by Roche (1 mentions)
Pepstatin, by Roche (1 mentions)
Complete edta free, by Roche (1 mentions)
Random hexamer primers, by NZYTech (1 mentions)
Nzydna ladder 6, by NZYTech (1 mentions)
Nzytaq green master mix, by NZYTech (1 mentions)
Dntps, by NZYTech (1 mentions)
Corticosterone, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
11 protocols using nzycolour protein marker 2
1
Optimizing Recombinant Protein Expression in E. coli
2
Phosphoproteomic Analysis of Maize Leaves
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All reagents used in this study are analytical or HPLC/MS grade. PhosStop, cOmplete EDTA-free, and pepstatin were acquired from Roche Applied Science (Germany). Nuclease Mix, Destreak Reagent, IPG Buffer pH 4–7, and 7 cm Immobiline® Drystrips pH 4–7 were obtained from GE Healthcare (UK). The phosphoprotein stain Pro-Q® Diamond (PQD) and the PeppermintStick™ phosphoprotein molecular weight markers were purchased from Life Technologies (CA, USA). The whole proteome Coomassie Brilliant Blue stain, BlueSafe (CBB) and the protein molecular weight markers NZYColour Protein Marker II were acquired from NZYTech (Portugal). Porcine trypsin was acquired from Promega Corporation (WI, USA). Seeds from Zea mays inbred line B73 used in this study were amplified in our greenhouse over the years. Original seeds were kindly provided by Dr. Christoph Peterhansel (University of Hannover, Germany).
3
Cellular Protein Quantification and Analysis
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Fetal bovine serum (FBS) was obtained from Merck-Millipore (Berlin, Germany). Insulin–transferrin–sodium selenite (ITS) supplement was purchased from Life Technolgies (Gaithersburg, MD, USA). Pierce BCA protein assay kit was obtained from Thermo Scientific (Waltham, MA, USA). Clarity™ western ECL substrate was purchased from Bio-Rad (Bio-Rad, CA, USA). NZYColour Protein Marker II, NZY M-MuLV reverse transcriptase (M-MLV RT), random hexamer primers, dNTPs, NZYTaq green master mix, Greensafe and NZY qPCR green master mix, and NZYDNA Ladder VI were obtained from NZYTech (Lisbon, Portugal). Corticosterone (with 98.5% purity minimum) and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other specific reagents are described alongside their respective methods.
4
Optimized Western Blot Protocol
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Samples for western blot were homogenized by a tissue lyser in
radioimmunoprecipitation assay (RIPA) buffer containing complete protease
inhibitor cocktail (Merck, Darmstadt, Germany) then centrifuged at
10,000 × g for 10 min to remove cellular debris.
Equivalent amounts of protein (20 µg) and the protein marker (NZYColour Protein
Marker II, MB090, NZYtech, Lisbon, Portugal) were separated on 12% acrylamide
gels (TGX Stain-Free™ FastCast™ Acrylamide kit; Bio-Rad, Hercules, CA, USA)
gels. This technology contains trihalo compounds that react with tryptophan
residues in a UV-induced reaction to produce fluorescence that can be detected
by the ChemiDoc imaging system. Total protein was detected using the ChemiDoc
imager in the membrane after transfer onto nitrocellulose membranes. Membranes
were blocked for 1 h with bovine serum albumin (BSA) 5% and then incubated with
primary antibody [H3K27me from Cell Signaling (9733) at 1:1000 dilution in 5%
BSA; Cell Signalling Technology, Beverly, MA, USA]. The membranes were incubated
for 1 h at room temperature with secondary antibody horseradish
peroxidase-conjugated anti-rabbit (1:1000). Detection was conducted using
SuperSignal West Femto chemiluminescent substrate kits (Pierce Biotechnology
Inc., Rockford, IL, USA) using the ChemiDoc imager.
radioimmunoprecipitation assay (RIPA) buffer containing complete protease
inhibitor cocktail (Merck, Darmstadt, Germany) then centrifuged at
10,000 × g for 10 min to remove cellular debris.
Equivalent amounts of protein (20 µg) and the protein marker (NZYColour Protein
Marker II, MB090, NZYtech, Lisbon, Portugal) were separated on 12% acrylamide
gels (TGX Stain-Free™ FastCast™ Acrylamide kit; Bio-Rad, Hercules, CA, USA)
gels. This technology contains trihalo compounds that react with tryptophan
residues in a UV-induced reaction to produce fluorescence that can be detected
by the ChemiDoc imaging system. Total protein was detected using the ChemiDoc
imager in the membrane after transfer onto nitrocellulose membranes. Membranes
were blocked for 1 h with bovine serum albumin (BSA) 5% and then incubated with
primary antibody [H3K27me from Cell Signaling (9733) at 1:1000 dilution in 5%
BSA; Cell Signalling Technology, Beverly, MA, USA]. The membranes were incubated
for 1 h at room temperature with secondary antibody horseradish
peroxidase-conjugated anti-rabbit (1:1000). Detection was conducted using
SuperSignal West Femto chemiluminescent substrate kits (Pierce Biotechnology
Inc., Rockford, IL, USA) using the ChemiDoc imager.
5
Comprehensive Protein Extraction and Separation
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All reagents used were HPLC grade or electrophoresis grade. Albumin, from bovine serum (BSA), urea, thiourea, 3-[(3-Cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS), β-mercaptoethanol, glycerol 86-88%, bradford reagent, coomassie blue R-250 (CBB), DL-dithiotreitol (DTT), iodoacetamide (IAA), trypsin sequencing trifluoroacetic acid (TFA), sodium deoxycholate (DOC) and acrylamide/bis-acrylamide 30% solution (37.5:1) were purchased from Sigma-Aldrich (St. Louis, USA). (N, N, N’, N’–tetramethylethylene-diamine (TMED), ammonium persulphate (APS), glycine were purchased from NZYTech (Lisbon, Portugal). Ampholytes pH 3–10, formic acid, and ammonium bicarbonate were purchased from Fluka (Steinheim, Germany). Hydrochloride acid (HCl), glacial acetic acid, tris-base, trichloroacetic acid (TCA), sodium dodecyl sulfate (SDS), methanol, acetonitrile were purchased from Panreac (Barcelona, Spain). Bromophenol blue was from Riedel-de Haën. Agarose and Mineral oil were purchased from Biorad (Hercules, USA).
Molecular weight marker for gel electrophoresis NZYColour Protein Marker II was purchased from NZYTech (Lisbon, Portugal). α-Cyano-4-hydroxycinnamic acid puriss for MALDI-MS from Fluka was used as MALDI matrix.
Molecular weight marker for gel electrophoresis NZYColour Protein Marker II was purchased from NZYTech (Lisbon, Portugal). α-Cyano-4-hydroxycinnamic acid puriss for MALDI-MS from Fluka was used as MALDI matrix.
6
SDS-PAGE Analysis of Codfish Protein
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the protein extracts obtained after SBW process of codfish frames was conducted by the method of Laemmli [46 (link)], with some modifications, to separate the proteins by their molecular weight. The hydrolysates, 100 μg protein of each sample, were mixed with the loading buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS (w/v), 10% glycerol (v/v), and 0.01% (w/v) bromophenol blue), and then loaded onto two different polyacrylamide gels: (i) a 7.5% precast gel, and (ii) a 15% resolving gel and 4.5% stacking gel. The electrophoresis was conducted at a constant voltage of 120 V. After electrophoresis, the gel was stained with 0.25% (w/v) Coomassie Blue R-250 in 8% (v/v) acetic acid and 46% (v/v) ethanol for 30 min and subsequently destained with a solution containing 10% (v/v) acetic acid and 30% (v/v) ethanol. A molecular-weight protein marker (NZYColour Protein Marker II) (NZYTech, Lda.—Genes and Enzymes, Lisbon, Portugal) was used to estimate the mass of the protein bands.
7
Preparation of High-Purity Reagents for Biochemical Assays
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Ultrapure reagent-grade water was obtained from a Milli-Q system (Millipore/Waters, Etten-Leur, The Netherlands). Ammonium sulfate, ethylenediamine tetra-acetic acid (EDTA) and sodium dodecyl sulfate (SDS) were obtained from PanReac Applichem (Darmstadt, Germany). Hydrochloric acid and Tween-20 were purchased from ThermoFisher Scientific (Waltham, MA, USA). Sodium chloride was obtained from Honeywell (Charlotte, NC, USA) Sodium deoxycholate was acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). Tris-base was obtained from Fisher Scientific (Epson, UK). Nonidet P-40 (NP-40) was purchased from Fluka (Monte Carlo, Monaco). Bis-Acrylamide/Acrylamide 40% was bought from GRiSP Research Solutions (Oporto, Portugal). The NZYColour Protein Marker II was acquired from NZYTech (Lisbon, Portugal). All chemicals were of analytical-grade commercially available and used without further purification.
8
Fractionation and Immunoblotting of Gliadin
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The different alcohol-soluble fractions of the commercial gliadin and the gluten-containing cereals were separated in two identical acrylamide gels (AnyKD precast-gel from Bio-Rad©, Hercules, CA, USA, #ref 4569033) under denaturing conditions at 90 V using NZYcolour Protein Marker II (NZYtech©, #ref M090). After electrophoresis, one of the gels was stained with Coomassie blue and the protein bands of the twin gel were transferred to a PVDF membrane at 100 V for 1.5 h. The PVDF membrane was blocked overnight with 3% BSA in TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.5) before incubation with a 1:45 dilution (TBS-BSA 1%) of the PEG-NaCl precipitated phage-Fab8E-4 for 2 h at 37 °C in a rocking platform. Membrane was rinsed 5 times with TBS and incubated for 1 h at 37 °C with the anti-M13proteinVIII-HRP secondary antibody diluted 1:5000 in blocking solution. The image was revealed after the addition of a chemo-luminescent reactive (Clarity™ ECL Western blotting substrate from Bio-Rad©, #ref 1705061) in a ChemiDoc Imaging System (Bio-Rad©).
9
Fosmid Library Production and Xylanase Purification
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The CopyControl™ Fosmid Library Production kit, including the autoinduction solution (500 ×), and the FosmidMAX™ DNA Purification kit were provided by Epicentre Biotechnologies (Madison, Wisconsin, USA). The NucleoSpin® Gel and PCR Clean-up kit and the NucleoSpin® Plasmid QuickPure™ kit were obtained from Macherey-Nagel (Düren, Germany). Azurin-crosslinked xylan (AZCL-xylan) was supplied by Megazyme (Wicklow, Ireland) and xylan from beechwood was obtained from Carl Roth (Karlsruhe, Germany). Congo red was acquired from HIMEDIA Laboratories Pvt. Ltd. (Maharashtra, India). NdeI and BamHI restriction enzymes and T4 DNA ligase were provided by Thermo Fisher Scientific (Waltham, Massachusetts, USA). pETDuet-1 expression vector was supplied by Novogene (Cambridge, UK). E. coli NZY5α, E. coli BL21 (DE3), bovine serum albumin (BSA), and NZYColour Protein Marker II were delivered by NzyTech (Lisboa, Portugal). Xylanase from Thermomyces lanuginosus, lipase from Candida rugosa, and other assay reagents used in this work (analytical grade) were purchased from Sigma-Aldrich (St. Louis, IL, USA).
10
Western Blot Analysis of Proteins
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Western blot was performed as described previously [13 (link)]. Briefly, total (25 µg), cytoplasmic (25 µg) or nuclear (30 µg) proteins were separated by SDS-PAGE under reducing conditions. A commercial mixture of 12 purified pre-stained proteins (NZYColour Protein Marker II, NZYTech, Lisbon, Portugal) was run in each gel to allow for confirmation of the apparent molecular weight of the proteins of interest. The proteins were then electrotransferred onto PVDF membranes (Immobilon®—P, Merck Millipore Ltd.) which were probed overnight at 4 °C or for 2 h at room temperature with the primary antibodies indicated in Table 1 and then with anti-rabbit (dilution 1:20,000; NIF1317, lot9465473, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20,000; NIF1316, lot6963606, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Mouse monoclonal anti-β-Tubulin I and rabbit polyclonal anti-Lamin B1 were used as a loading controls of total and cytoplasmic extracts and of nuclear extracts, respectively. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system ThyphoonTM FLA 9000 (GE Healthcare). Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
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