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Sypro ruby protein gel stain

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SYPRO Ruby protein gel stain is a fluorescent stain used for the detection of proteins in polyacrylamide gels. It is a sensitive stain that can detect as little as 1 ng of protein per band.

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Lab products found in correlation

Typhoon 9410, by GE Healthcare (3 mentions) Bis trispolyacrylamide gels, by Bio-Rad (2 mentions) Image lab 6, by Bio-Rad (2 mentions) Lds loading buffer, by Thermo Fisher Scientific (2 mentions) Heat inactivated bovine serum, by Thermo Fisher Scientific (2 mentions) Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions) Gel doc xr, by Bio-Rad (2 mentions) Native gel sample buffer, by Thermo Fisher Scientific (2 mentions) Tris glycine gel, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4 fast flow resin, by GE Healthcare (2 mentions) Ecl plus western blotting substrate, by Bio-Rad (1 mentions) Phenol red, by Lonza (1 mentions) Qev size exclusion column, by Izon Science (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Odyssey clx infrared imaging system, by LI COR (1 mentions) Image studio imaging software, by LI COR (1 mentions) Protein assay, by Bio-Rad (1 mentions) Quantity one 4, by Bio-Rad (1 mentions) Tgx gel, by Bio-Rad (1 mentions) Pvdf membrane, by Thermo Fisher Scientific (1 mentions)

16 protocols using sypro ruby protein gel stain

1

Comparative Proteomic Analysis of Stem/Progenitor Cells

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Equal numbers of cells from each stem or progenitor population were sorted into HBSS supplemented with 2% (v/v) heat-inactivated bovine serum (GIBCO). Samples were washed once with PBS and centrifuged at 2500 g at 4°C for 5 min. Supernatant was discarded and protein pellets were directly lysed and solubilized in 9M urea, 2% SDS, 50 mM DTT, and 50 mM Tris pH 7.4. After incubating the samples for 10–15 min at room temperature, LDS loading buffer (Life Technologies) was added and the sample was heated at 70°C for 10 minutes. Samples were separated on Bis-Trispolyacrylamide gels (Life Technologies). The Bis-Trispolyacrylamide gels were then stained with SYPRO Ruby Protein Gel Stain (Bio-Rad) for 3 h and washed in a solution containing 7% acetic acid and 10% methanol for 1 h. Gels were imaged using Gel Doc XR (Bio-Rad) and analyzed with Image Lab 6.0.1 software (Bio-Rad).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
Jose L.H., Sunshine M.J., Dillingham C.H., Chua B.A., Kruta M., Hong Y., Hatters D.M, & Signer R.A. (2020). Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal. Cell reports, 30(1), 69-80.e6.
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2

Native PAGE Analysis of NLP Samples

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Equal mass aliquots of NLP samples (1.5 μg) were diluted with 2x native gel sample buffer (Invitrogen) and loaded onto 4–12% gradient pre-made Tris-glycine gels (Invitrogen). Samples were electrophoresed for 2 hrs. at a constant 125 V. After electrophoresis, gels were incubated with SYPRO Ruby protein gel stain (Bio-Rad) for 2 hours and then de-stained using 10% Methanol, 7% Acetic acid. Following a brief wash with ddH2O, gels were imaged using the green laser (532 nm) of a Typhoon 9410 (GE Healthcare) with a 610 nm bandpass 30 filter. Molecular weights were determined by comparing migration vs. log molecular weight of standard proteins found in the NativeMark standard (Invitrogen).
Coleman M.A., Cappuccio J.A., Blanchette C.D., Gao T., Arroyo E.S., Hinz A.K., Bourguet F.A., Segelke B., Hoeprich P.D., Huser T., Laurence T.A., Motin V.L, & Chromy B.A. (2016). Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles. PLoS ONE, 11(3), e0150166.
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3

Protease-mediated Degradation of rIL-23R

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The full-length human recombinant interleukin-23 receptor (rIL-23R, 10 nM) (Abnova, Jhouzih St. Taipei, Taiwan), embedded in proteoliposomes, was incubated at 37 °C in the presence or absence of Der p 1 (2.5 nM, preactivated with 1 mM DTT), Der p 3 (0.25 nM) or Der p 6 (0.5 nM) in PBS pH 7.5 for different periods of time (from 0 to 24 h). At the appropriate time points, proteolysis was stopped by the addition of protease inhibitors (10 µM E-64 or PMSF). rIL-23R degradation was analyzed by SDS-PAGE and revealed using the Sypro Ruby protein gel stain (Bio-Rad, Temse, Belgium) and by western blot analysis using a rabbit anti-human IL-23R (residues 62–75) monoclonal primary antibody (SAB1104999, dilution 1/1000) (Sigma-Aldrich) and a goat HRP/anti-rabbit IgG monoclonal conjugate as secondary antibody (1706515, dilution 1:3000) revealed using the ECL plus western blotting substrate (Bio-Rad).
Jacquet A., Campisi V., Szpakowska M., Dumez M.E., Galleni M, & Chevigné A. (2017). Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates. International Journal of Molecular Sciences, 18(7), 1373.
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4

Characterization of ZIKV NS5-hSTAT2 Interaction

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60 μg of GST-hSTAT2 fusion protein was incubated with 150 μg of ZIKV NS5, in the form of either full length or individual domains, wild type or mutants, in a 1 mL pull-down buffer (10 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% glycerol, 5mM DTT and 0.1% Triton X-100) for 1 h at 4°C. The mixtures were centrifuged at the speed of 15,000 rpm on an 5424R Eppendorf microcentrifuge for 15 min, followed by incubation of the supernatant with 20 μL glutathione sepharose 4 fast flow resins (GE Healthcare) for 15 min. Subsequently, the beads were separated from the supernatant through centrifugation at the speed of 500 rpm and subjected to two and four washes with the pull-down buffer for NS5 mutants and hSTAT2 mutants, respectively. The bound proteins were analyzed by SDS–PAGE followed by SYPRO Ruby protein gel stain (Bio-Rad). For IRF9 competition assay, the mixture of GST-hSTAT2 and ZIKV NS5 was further incubated with IRF9 in an IRF9:NS5 molar ratio of 1:40, 1:20 or 1:10, followed by washes and SDS-PAGE analyses as described above.
Wang B., Thurmond S., Zhou K., Sánchez-Aparicio M.T., Fang J., Lu J., Gao L., Ren W., Cui Y., Veit E.C., Hong H., Evans M.J., O’Leary S.E., García-Sastre A., Zhou Z.H., Hai R, & Song J. (2020). Structural basis for STAT2 suppression by flavivirus NS5. Nature structural & molecular biology, 27(10), 875-885.
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5

Fractionation of Raw Cow Milk Proteins

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To fractionate raw cow’s milk based on molecular size, qEV size exclusion columns (Izon Science, Oxford, UK) were used according to the manufacturer’s protocol. Briefly, 0.5 mL of 10,000× g raw milk supernatant (free of cells, cell debris, and cream) was loaded onto the size exclusion column (Izon Science) and the first 3 mL of eluent was discarded. Eluent fractions of 0.5 mL were then collected up to 12 mL (24 fractions), by continuously adding RPMI 1640 medium without l-glutamine and phenol red (Lonza) to the column. Protein content of each fraction was quantified by using a NanoDrop ND-1000 spectrophotometer (A280; Thermo Fisher Scientific). To determine the molecular weight of the proteins in each fraction, proteins were separated by using a 12.5% SDS-PAGE under non-reducing conditions and visualized with SYPRO® Ruby Protein Gel Stain (Bio-Rad, Veenendaal, The Netherlands). Fractions were stored at −80 °C until further use.
Abbring S., Blokhuis B.R., Miltenburg J.L., Olmedo K.G., Garssen J., Redegeld F.A, & van Esch B.C. (2020). Direct Inhibition of the Allergic Effector Response by Raw Cow’s Milk—An Extensive In Vitro Assessment. Cells, 9(5), 1258.
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6

SLO Detection via Western Blot

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CSPs were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham; 0.45 μm). For the detection of SLO, a monoclonal anti-streptolysin O antibody (Abcam) was diluted 1:5,000 in 5% nonfat milk and incubated with the membranes for 1 h at room temperature. The membranes were then washed with a solution of phosphate-buffered saline (PBS)–Tween (0.1%) 3 times for 5 min each. The primary antibody was detected using an anti-mouse IgG secondary antibody conjugated with IRDye 700 dye (Li-Cor), which was diluted 1:10,000 in 5% nonfat milk and incubated with the membrane blots for 1 h. The blots were washed and analyzed with densitometry using an Odyssey CLx infrared imaging system with Image Studio imaging software (Li-Cor). Densitometry values (expressed in arbitrary units [AU]) were obtained using the Image Studio imaging software associated with the Li-Cor Odyssey infrared imaging system. To confirm that equal amounts of protein were analyzed, a separate SDS-PAGE gel was stained for total protein using Sypro ruby protein gel stain (Bio-Rad).
Herrera A.L., Callegari E.A, & Chaussee M.S. (2021). The Streptococcus pyogenes Signaling Peptide SpoV Regulates Streptolysin O and Enhances Survival in Murine Blood. Journal of Bacteriology, 203(11), e00586-20.
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7

Radiolabeling of Huh7 Cells Expressing tRH Variants

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Huh7 cells were seeded onto the wells of 6-well cell culture plates at a density of 2 × 105 cells/well and incubated overnight to allow cell attachment. Cells were transfected with 50 nM and 100 nM of 5′ tRHGly (5′-GCAUUGGUGGUUCAGUGGUAGAAUUCUCGCCU-3′), 5′ tRHVal (5′-GUUUCCGUAGUGUAGUGGUUAUCACGUUCGCCU-3′), or scramble (5′-GCAUUCACUUGGAUAGUAAAUCCAAGCUGAA-3′)21 (link) (all from Integrated DNA Technologies, Coralville, IA) oligonucleotide after replacing cell culture medium with methionine- and cysteine-deficient DMEM (Life Technologies) and cultured for further 12 hrs. Cells were then metabolically radiolabeled for 12 hrs with 200 μCi/well of Express Protein Labeling Mix containing [35S]methionine and [35S]cysteine (PerkinElmer, Waltham, MA) in the presence or absence of 50 μg/ml puromycin and lysed with lysis buffer (20 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 1% Triton X-100, 0.05% SDS, and 10% glycerol) supplemented with 50 mM NaF, 5 mM Na3VO4, and a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). The protein concentration of cell lysates was determined by the Bio-Rad Protein Assay (Bio-Rad), and 10 μg (total protein) of cell lysates was subjected to SDS-PAGE followed by staining gels with the Sypro Ruby Protein Gel Stain (Bio-Rad, Hercules, CA) and autoradiography.
Selitsky S.R., Baran-Gale J., Honda M., Yamane D., Masaki T., Fannin E.E., Guerra B., Shirasaki T., Shimakami T., Kaneko S., Lanford R.E., Lemon S.M, & Sethupathy P. (2015). Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C. Scientific Reports, 5, 7675.
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8

Comparative Proteomic Analysis of Stem/Progenitor Cells

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Equal numbers of cells from each stem or progenitor population were sorted into HBSS supplemented with 2% (v/v) heat-inactivated bovine serum (GIBCO). Samples were washed once with PBS and centrifuged at 2500 g at 4°C for 5 min. Supernatant was discarded and protein pellets were directly lysed and solubilized in 9M urea, 2% SDS, 50 mM DTT, and 50 mM Tris pH 7.4. After incubating the samples for 10–15 min at room temperature, LDS loading buffer (Life Technologies) was added and the sample was heated at 70°C for 10 minutes. Samples were separated on Bis-Trispolyacrylamide gels (Life Technologies). The Bis-Trispolyacrylamide gels were then stained with SYPRO Ruby Protein Gel Stain (Bio-Rad) for 3 h and washed in a solution containing 7% acetic acid and 10% methanol for 1 h. Gels were imaged using Gel Doc XR (Bio-Rad) and analyzed with Image Lab 6.0.1 software (Bio-Rad).
For bone marrow cells, incubated at 37°C or 42°C, 105 cells were sorted into TCA. The final concentration was adjusted to 10% TCA. Extracts were incubated on ice for at least 15 min and centrifuged at 15,000 g at 4°C for 15 min. Precipitates were washed in acetone twice and dried. The pellets were solubilized in 6 M urea, 2% (v/v) Triton X-100, and 1% (v/v) 2-mercaptoethanol. The protein content in the supernatant was assessed with the microBCA assay (Pierce).
Jose L.H., Sunshine M.J., Dillingham C.H., Chua B.A., Kruta M., Hong Y., Hatters D.M, & Signer R.A. (2020). Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal. Cell reports, 30(1), 69-80.e6.
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9

Gel-Based Protein Expression Analysis

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For image analysis, gels were stained with fluorescent SYPRO Ruby Protein Gel Stain (Bio‐Rad) according to the manufacturer's instructions. Fluorescent stained gels were digitalized in a Molecular Imager FX scanner, obtaining images with Quantity One 4.2.3 software (Bio‐Rad) and analyzing them with ProgenesisSameSpots software (Nonlinear Dynamics, Newcastle, UK). The gel patterns from each independent analysis were matched and the relative volume of each spot (%V) in the two gel sets were compared (disease‐free and metastasis progression). Spots were considered to be expressed distinctly if the difference in the relative volume of each spot was significant at p < 0.05. Gels were stored in deionized water at 4°C and to visualize the spots of interest for further analysis, silver staining was performed as described by Rabilloud,11 with minor modifications.
Ortega‐Martínez I., Gardeazabal J., Erramuzpe A., Sanchez‐Diez A., Cortés J., García‐Vázquez M.D., Pérez‐Yarza G., Izu R., Luís Díaz‐Ramón J., de la Fuente I.M., Asumendi A, & Boyano M.D. (2016). Vitronectin and dermcidin serum levels predict the metastatic progression of AJCC I–II early‐stage melanoma. International Journal of Cancer, 139(7), 1598-1607.
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10

Characterization of ZIKV NS5-hSTAT2 Interaction

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60 μg of GST-hSTAT2 fusion protein was incubated with 150 μg of ZIKV NS5, in the form of either full length or individual domains, wild type or mutants, in a 1 mL pull-down buffer (10 mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% glycerol, 5mM DTT and 0.1% Triton X-100) for 1 h at 4°C. The mixtures were centrifuged at the speed of 15,000 rpm on an 5424R Eppendorf microcentrifuge for 15 min, followed by incubation of the supernatant with 20 μL glutathione sepharose 4 fast flow resins (GE Healthcare) for 15 min. Subsequently, the beads were separated from the supernatant through centrifugation at the speed of 500 rpm and subjected to two and four washes with the pull-down buffer for NS5 mutants and hSTAT2 mutants, respectively. The bound proteins were analyzed by SDS–PAGE followed by SYPRO Ruby protein gel stain (Bio-Rad). For IRF9 competition assay, the mixture of GST-hSTAT2 and ZIKV NS5 was further incubated with IRF9 in an IRF9:NS5 molar ratio of 1:40, 1:20 or 1:10, followed by washes and SDS-PAGE analyses as described above.
Wang B., Thurmond S., Zhou K., Sánchez-Aparicio M.T., Fang J., Lu J., Gao L., Ren W., Cui Y., Veit E.C., Hong H., Evans M.J., O’Leary S.E., García-Sastre A., Zhou Z.H., Hai R, & Song J. (2020). Structural basis for STAT2 suppression by flavivirus NS5. Nature structural & molecular biology, 27(10), 875-885.
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