Readyblue protein gel stain

Manufactured by Merck Group

Sourced in Germany, United States

ReadyBlue Protein Gel Stain is a ready-to-use, pre-mixed staining solution for the detection of proteins in polyacrylamide gels. It provides a simple and efficient method for visualizing protein bands after electrophoresis.

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10 protocols using readyblue protein gel stain

Native Protein Separation and Oligomeric State Determination

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The hrCN PAGE protocol was adapted from Lemaire et al. (2018) (link). Glycerol was added to the sample at a final amount of 20% (v/v). Ponceau S at a final concentration of 0.001% (w/v) served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM tricine, 15 mM Bis-Tris/HCl, pH 7.0, 0.05% (w/v) sodium deoxycholate, 2 mM DTT, and 0.01% (w/v) dodecyl maltoside, whilst the anode buffer contained 50 mM Bis-Tris/HCl, PH 7.0, 2 mM DTT. An 8–15% linear polyacrylamide gradient gel was used, and electrophoresis was run under a N₂/CO₂ (90:10%) atmosphere with a constant 40 mA current (PowerPac^™ Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^™ Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^™ Unstained Protein Standard (Thermo Fischer Scientific, Driesch, Germany).
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N₂/H₂ (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.

Lemaire O.N., Belhamri M, & Wagner T. (2023). Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon. Frontiers in Microbiology, 14, 1179204.

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SDS-PAGE and Western Blot Protocol

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Samples were mixed with sample loading buffer (Licor 928-40004) supplemented with DTT and incubated at 95° for 5 minutes. SDS-PAGE was performed with pre-cast 4–12% gradient gels (ThermoFisher NW04127BOX) in MOPS (ThermoFisher B000102) according to the manufacturer’s instructions.
For Coomassie staining, gels were washed thoroughly in water, incubated with ReadyBlue Protein Gel Stain (Sigma RSB-1L) overnight, and destained in water. For western blots, proteins were transferred to nitrocellulose membranes by semi-dry transfer (Biorad 1704158) according to the manufacturer’s instructions. Membranes were rinsed in water and stained with Revert 700 Total Protein Stain according to the manufacturer’s instructions. The membranes were then rinsed in TBS, rocked with Everyblot Blocking Buffer (Biorad 12010020) at room temperature for > 30 minutes, and rocked with primary antibody overnight at 4°. The membranes were washed with TBST, and rocked with IR800CW-labeled secondary antibodies for 30–60 minutes, washed with TBST, and imaged on a Licor Odyssey CLx.

Replogle J.M., Saunders R.A., Pogson A.N., Hussmann J.A., Lenail A., Guna A., Mascibroda L., Wagner E.J., Adelman K., Lithwick-Yanai G., Iremadze N., Oberstrass F., Lipson D., Bonnar J.L., Jost M., Norman T.M, & Weissman J.S. (2022). Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Cell, 185(14), 2559-2575.e28.

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Protein Characterization via SDS-PAGE and Mass Spectrometry

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The concentration of purified proteins was determined using NanoDrop
One (Thermo Scientific). For performing SDS-PAGE, protein samples
were set to contain 0.1, 0.5, 1, and 2 μg, mixed with the Laemmli
buffer, and incubated at 95 °C for 15 min. After incubation,
the samples were spun down and 20 μL was loaded on a pre-cast
12% agarose gel (MiniProtean TGX gel, BIO-RAD). SDS-PAGE was performed
at 130 V for 40 min. Afterward, gels were stained for 1 h using ReadyBlue
Protein Gel Stain (Sigma-Aldrich) and destained with ddH₂O overnight. Images were taken with the ChemiDoc XRS+ system (BIO-RAD).
Protein bands analyzed using mass spectrometry were excised, treated
with 10 mM dithiothreitol and 10 mM iodoacetamide, and subsequently
digested with trypsin. The peptide solution was then separated on
a Waters Acquity I-class UPLC in positive HD-MSE mode using a Waters
Peptide CSH C18 column (2.1 mm × 150 mm, 1.7 μm particle
size). A gradient from 1 to 40% ACN/0.1% formic acid (v/v) in water/0.1%
formic acid (v/v) was utilized at a starting temperature of 80 °C
and a dissolving temperature of 400 °C with a gas flow rate of
800 l/h. Spectra obtained by the separation were analyzed by matching
with the UniProt database.

Wehler P., Armbruster D., Günter A., Schleicher E., Di Ventura B, & Öztürk M.A. (2022). Experimental Characterization of In Silico Red-Shift-Predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S Mutants. ACS Omega, 7(23), 19555-19560.

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Investigating E. coli BL21 protein expression

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E. coli BL21 were purchased from Invitrogen. Plasmid pET-21A was purchased from Novagen. pDNR-LIR-hVDAC was purchased from Dharmacon. Ampicillin, IPTG, Triton 100X, Guanidine hydrochloride, Acrylamide: Bisacrylamide 37.5:1, SDS 10%, Temed, APS, TGS 10X, NH₄Cl and CaCl₂ were purchased from Euromedex. NaCl, NaH₂PO₄, K₂HPO₄, KH₂PO₄, glucose, LB-broth, Tris-HCl, NADH, propofol, quinidine, ubiquinone 0, Laemli 6X, β-mercapto ethanol, PBS, Na₂HPO₄, K₂SO₄ and metformin were purchased from ThermoFisher. Aspirin, catechin, curcumin, DCCD, DPC, emodin, fluoxetine, and MnCl₂ were purchased from Fluorochem. Imidazole, DIDS, DMSO, ReadyBlue Protein Gel stain, EDTA, MgCl₂, CoCl₂, H₂BO₃, acetone and ethanol were purchased from Sigma-Aldrich. FeSO₄ and ZnSo₄ were purchased from Biobasic. CuCl₂ and Mo₇O₂₄ were purchased from Roth. ¹⁵NH₄Cl, D₂O and deuterated glucose were purchased from Innova-Chem. LDAO was purchased from CliniSciences. Itraconazole was purchased from Cayman Chemical company. VBIT4 was purchased from Aobious. Cannabidiol was purchased from Carbosynth. Olesoxime was obtained from in-house chemical library.

Gorny H., Mularoni A., Delcros J.G., Freton C., Preto J, & Krimm I. (2023). Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules. Journal of Enzyme Inhibition and Medicinal Chemistry, 38(1), 2121821.

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Protein Separation and Quantification

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For both native and SDS-PAGE, 4-15% Mini-PROTEAN TGX gels (Bio-Rad) were used. 10x running buffer consisted of 250 mM Tris, 1.92 M glycine for native PAGE and 1% (w/v) SDS was added to the SDS-PAGE running buffer. 2x native sample buffer consisted of 62.5 mM Tris-HCl, 40% (v/v) glycerol, 0.01% bromophenol blue. As a denaturating sample buffer, 2x Laemmli buffer with 10% 2-mercaptoethanol was used. Gels were run at 100-120 V for 60 min in a Mini-PROTEAN Tetra Cell (Bio-Rad) and stained with ReadyBlue™ Protein Gel Stain (Sigma-Aldrich) for 1 h. Gel densitometry analysis was performed in ImageJ 1.50e.

Schastnaya E., Raguz Nakic Z., Gruber C.H., Doubleday P.F., Krishnan A., Johns N.I., Park J., Wang H.H, & Sauer U. (2021). Extensive regulation of enzyme activity by phosphorylation in Escherichia coli. Nature Communications, 12, 5650.

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Denaturing Gel Electrophoresis of Ab-MSNA Conjugates

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Denaturating polyacrylamide
gel electrophoresis was used to analyze the Ab–MSNA conjugates.
A precast gel cover (4–15% Mini-PROTEAN TGX Precast Protein,
Bio-Rad) was fixed into a vertical electrophoresis chamber, and the
running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3)
was filled into the chamber. Samples (mixed with 4× Laemmli Sample
buffer, 10% 2-mercaptoethanol) and a protein ladder (Precision Plus
Protein Dual Color Standard, Bio-Rad) were loaded and electrophoresed
at 200 V for approximately 30 min. After completion of electrophoresis,
gel was removed from the chamber and stained with Readyblue Protein
Gel Stain (Sigma-Aldrich) for 2 h prior to imaging with a Gel Doc
imaging system (Bio-Rad).

Äärelä A., Räsänen K., Holm P., Salo H, & Virta P. (2023). Synthesis of Site-Specific Antibody–[60]Fullerene–Oligonucleotide Conjugates for Cellular Targeting. ACS Applied Bio Materials, 6(8), 3189-3198.

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SDS-PAGE and Western Blot Protocol

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Purified KpDyP Protein Analysis

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Purified free KpDyP was subjected to size exclusion analysis on a Superdex 200 Increase 10/300 GL column. Fractions were then analyzed via native PAGE in an Invitrogen XCell SureLock using NativePAGE 3–12% bis-tris mini protein gels, 1X NativePAGE Running Buffer, and NativeMark Unstained Protein Standard from Fisher Scientific (USA) with 1× running buffer made from 10× Tris/Glycine Buffer from Bio-Rad Laboratories, Inc. (USA). Approximately 10 μg of protein was loaded per well. NativePAGE gels were run at 150 V for 1 h, then 250 V for 2.5 h at 4 °C. Gels were stained with ReadyBlue Protein Gel Stain from Sigma-Aldrich (USA) and imaged and analyzed on a ChemiDoc Imaging System by Bio-Rad Laboratories, Inc. (USA).

Jones J.A., Andreas M.P, & Giessen T.W. (2024). Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae. Nature Communications, 15, 2558.

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SDS-PAGE Analysis of Protein Samples

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Protein samples for SDS-PAGE analysis were prepared by mixing with Invitrogen NuPAGE LDS Sample Buffer (4x) (Invitrogen, Cat# NP0007) containing 350 mM 2-mercaptoethanol and heat-denaturing for 2 minutes at 95 °C. The protein samples were then run on NuPAGE 4–12% Bis-Tris gels (Invitrogen, Cat# NP0323BOX) using Novex NuPAGE MES SDS running buffer at 200 V for 35 min. Gels were stained using ReadyBlue protein gel stain (Sigma-Aldrich, Cat# RSB-1L) or Coomassie Brilliant Blue R-250 (Thermo Scientific, Ref # 20278) followed by de-staining with water or de-staining buffer. Gels were imaged using a Bio-Rad Chemidoc Imaging station.

Andreas M.P, & Giessen T.W. (2024). Cyclodipeptide oxidase is an enzyme filament. Nature Communications, 15, 3574.

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Native-PAGE Enzyme Activity Assay

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Precast SDS-PAGE gels were run at 30 mA per gel for 1 h on a BioRad Ready Strip IPG/Protean II system. Gels were stained with ReadyBlue Protein Gel Stain (Sigma-Aldrich). Enzyme activity staining was performed with Native-PAGE gels and cytoplasmic cell fractions under anoxic conditions (52 (link)). P. fastidiosa phosphite-oxidizing activity was stained by immersing the gel strips in 25 mM HEPES buffer, pH 8.0, containing 3 mM MgCl₂, and D. phosphitoxidans phosphite-oxidizing activity in 25 mM MOPS buffer, pH 7.2, containing 3 mM MgCl₂ and 17 mM NaCl, each with addition of 2 mM AMP, 2 mM NAD⁺, 10 mM sodium phosphite, and 0.4 mM iodonitrotetrazolium chloride (INT) as the final electron acceptor in order to indirectly detect NADH formation. Protein bands excised from stained Native-PAGE gels and SDS-PAGE gels were identified by peptide mass fingerprinting at the Proteomics Facility of the University of Konstanz.

Mao Z., Fleming J.R., Mayans O., Frey J., Schleheck D., Schink B, & Müller N. (2023). AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation. Proceedings of the National Academy of Sciences of the United States of America, 120(45), e2309743120.

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