Sypro ruby

Manufactured by Bio-Rad

Sourced in United States

SYPRO Ruby is a fluorescent stain used for detecting proteins in polyacrylamide gels. It exhibits excitation and emission maxima of 290 nm and 610 nm, respectively, and is compatible with visible and ultraviolet (UV) light detection methods.

Automatically generated - may contain errors

Lab products found in correlation

Chemidoc mp, by Bio-Rad (6 mentions) Typhoon fla 9000, by GE Healthcare (3 mentions) Image lab software, by Bio-Rad (3 mentions) Enhanced chemiluminescence detection reagent, by Thermo Fisher Scientific (2 mentions) Molecular imager chemidoc xrs, by Bio-Rad (2 mentions) Bis tris gel, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Thermo Fisher Scientific (2 mentions) Quantity one software, by Bio-Rad (2 mentions) Ettan daltsix electrophoresis unit, by Cytiva (2 mentions) Versadoc mp 4000 imaging system, by Bio-Rad (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Prism 7.0a, by GraphPad (2 mentions) Chemidoc mp imaging system, by Bio-Rad (2 mentions) Readystrip ipg strip, by Bio-Rad (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Typhoon trio scanner, by GE Healthcare (1 mentions) Mini protean 2 electrophoresis cell, by Bio-Rad (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Sp sepharose fast flow matrix, by GE Healthcare (1 mentions)

56 protocols using sypro ruby

E1-E2 Thioester Transfer Assay

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

E1-E2 thioester transfer assay was adapted from the previously described protocol^{33 (link)}. Assays were performed with 20 nM E1 (species-matched to E2), 500 nM E2, 5 μM Ub, and 1 mM ATP in 20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl₂ buffer. Reaction was started by addition of ATP and mixing then quenched after 30 s by addition of 2x UREA sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer. Owing to inherently low activity, Ubc15 thioester transfer assay was conducted with 50 nM E1 for 60 s. Samples (7.5 μl each) were subjected to SDS-PAGE at 150 V. Gels were then stained with SYPRO Ruby (BioRad) and visualized with a ChemiDoc MP (BioRad). Data quantification was conducted using densitometry in ImageJ software with original unedited images. Densitometry measurements were normalized as a percentage of the control WT assay on the same gel. Data are represented as an average of 3–4 technical replicates with + /−1 standard deviation error bars. Unprocessed images of representative gels for all biochemical assays are provided in the Source Data file.

Williams K.M., Qie S., Atkison J.H., Salazar-Arango S., Alan Diehl J, & Olsen S.K. (2019). Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34. Nature Communications, 10, 3296.

+ Open protocol

+ Expand

Ubiquitin E1-E2 Thioester Transfer Assay

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

The E1-E2 thioester transfer assays were performed at 25°C as previously described (Olsen and Lima, 2013 (link)). For all experiments the Ub E1 ortholog corresponding to that of the E2 being tested (human, S. pombe, or S. cerevisiae) was used. For the comparison of S. pombe Ub E2 activities in Figure 1A, assays were performed with 2.5 nM E1, 500 nM E2, 5 µM Ub, 5 mM MgCl₂, 200 µM ATP, 20 mM Hepes pH 7.5, 50 mM NaCl and were incubated for 30 s at room temperature. For the endpoint assay of Ubc15 activity in Figure 1B, reactions were performed with 1 µM E1, 500 nM E2, 5 µM Ub, 5 mM MgCl₂, 200 µM ATP, 20 mM Hepes pH 7.5, 50 mM NaCl and were incubated for 5 min at room temperature. For all subsequent structure-function analysis throughout the paper, thioester transfer assays were performed with 2.5 nM E1, 500 nM E2, 5 µM Ub, 5 mM MgCl₂, 200 µM ATP, 20 mM Hepes pH 7.5, 50 mM NaCl and the incubation times were adjusted based on activity levels, in order to remain in the linear range. Incubation times were as follows: 150 s for Ubc15 and Ubc1, 600 s UBE2H, 20 min for hUBE2K, 30 s for all other E2s. Reactions were terminated though the addition of non-reducing Urea SDS-PAGE buffer and subjected to SDS-PAGE, 150 V constant, at 4°C. The gels were stained with Sypro Ruby (BioRad) and visualized with a ChemiDoc MP (BioRad).

Lv Z., Rickman K.A., Yuan L., Williams K., Selvam S.P., Woosley A.N., Howe P.H., Ogretmen B., Smogorzewska A, & Olsen S.K. (2017). S. pombe Uba1-Ubc15 structure reveals a novel regulatory mechanism of ubiquitin E2 activity. Molecular cell, 65(4), 699-714.e6.

+ Open protocol

+ Expand

HPRT Crosslinking with DMA Ligand

Check if the same lab product or an alternative is used in the 5 most similar protocols

Crosslinking was performed with ≈10 μM B. subtilis HPRT (measured by monomer), 20 mM dimethyl adipimidate (DMA) (Thermo Scientific), and 500 μM ligand. DMA was suspended in 25 mM HEPES pH 8.0, 100 mM NaCl, 10 mM MgCl₂, and 10% glycerol, and the solution was buffered to pH 8.5. HPRT was dialyzed into 25 mM HEPES pH 7.5, 100 mM NaCl, and 10% glycerol. Ligands were incubated with protein for 10 min followed by a 15-min incubation with DMA at room temperature. Reactions were terminated with addition of 2X Laemmli buffer (Bio-Rad) for immediate analysis with SDS-PAGE (10% polyacrylamide gel). Gels were stained with SYPRO Ruby (Bio-Rad) according to the manufacturer’s protocol and imaged using a Typhoon FLA9000 (GE Healthcare).

Anderson B.W., Liu K., Wolak C., Dubiel K., She F., Satyshur K.A., Keck J.L, & Wang J.D. (2019). Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction. eLife, 8, e47534.

+ Open protocol

+ Expand

Protein Separation and Identification by LC-MS/MS

Check if the same lab product or an alternative is used in the 5 most similar protocols

For LC–MS/MS analysis, protein samples were boiled for 5 min and resolved in 12.5% acrylamide gels, using a Mini-Protean II electrophoresis cell (Bio-Rad). A constant voltage of 150 V was applied for 45 min. Gels were fixed in a solution containing 10% acetic acid and 40% ethanol for 30 min and stained overnight in Sypro Ruby (Bio-Rad). Gels were then washed in a solution containing 10% ethanol and 7% acetic acid for 30 min, and the image was acquired using a Typhoon Trio scanner (GE Healthcare). Each lane was cut in slices and subjected to tryptic digestion, followed by LC–MS/MS analysis.

Hermanova I., Zúñiga-García P., Caro-Maldonado A., Fernandez-Ruiz S., Salvador F., Martín-Martín N., Zabala-Letona A., Nuñez-Olle M., Torrano V., Camacho L., Lizcano J.M., Talamillo A., Carreira S., Gurel B., Cortazar A.R., Guiu M., López J.I., Martinez-Romero A., Astobiza I., Valcarcel-Jimenez L., Lorente M., Arruabarrena-Aristorena A., Velasco G., Gomez-Muñoz A., Suárez-Cabrera C., Lodewijk I., Flores J.M., Sutherland J.D., Barrio R., de Bono J.S., Paramio J.M., Trka J., Graupera M., Gomis R.R, & Carracedo A. (2020). Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer. The Journal of Experimental Medicine, 217(6), e20191787.

+ Open protocol

+ Expand

Purification of N-terminal FLAG-tagged hTDP1

Check if the same lab product or an alternative is used in the 5 most similar protocols

N-terminal Flag-tagged hTDP1 alleles were expressed from YEpGal4-10GAL1-FLAGhTDP1·L vectors in protease-deficient top1Δ,tdp1Δ cells (ECY2 strain). Exponential cultures were induced with 2% galactose for 6 hours, cells were harvested, and FLAGhTDP1 protein purified as described in [32 (link)]. Briefly, cell extracts in HEE/2PI buffer (50 mM HEPES, pH 8.0, 5 mM EDTA, 5mM EGTA, 2PI [2xprotease inhibitor cocktail, Sigma]) were loaded onto SP Sepharose fast flow matrix (GE Life Sciences) and eluted with HEE/2PI/0.4 M NaCl, then affinity purified by anti-FLAG M2 affinity chromatography matrix (Sigma). FLAGhTDP1 was eluted with 3X FLAG peptide in TEEK (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM DTT) and concentrated in an Ultracel-30K concentrator (Millipore). Concentration was determined by Bradford-assay (Bio Rad) and purity was determined by sypro-ruby (Bio Rad) staining of hTDP1 fractions resolved by 12% SDS-PAGE.

Cuya S.M., Comeaux E.Q., Wanzeck K., Yoon K.J, & van Waardenburg R.C. (2016). Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin. Oncotarget, 7(52), 86660-86674.

+ Open protocol

+ Expand

Cardiac Protein Isolation and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Frozen mouse hearts were homogenized for total protein or myofilament-enriched fractions. All buffers used for protein isolation contained protease inhibitors (Roche 4693159001) and phosphatase inhibitors (Sigma P5726 and P0044). Proteins were separated by SDS-PAGE on either 10% polyacrylamide or 4-15% polyacrylamide gradient gels. Proteins were transferred to nitrocellulose membranes at 4°C at 300mA for 3 hours in Tris glycine with 10% methanol. Membrane blocking and antibody incubations were performed using Tris-buffered saline (TBS) with 1% Tween-20 with blocking buffer (Roche 11921681001). Antibodies recognizing cardiac-specific N’-terminal regions of cMyBP-C (Santa Cruz 137180), actin (Sigma A2172), and phospho cTnI (Cell Signaling 4004) were used for Western blot with anti-rabbit and anti-mouse secondary HRP-conjugated antibodies for detection (Santa Cruz SC-2004 and SC-2314). ECL Prime (GE Life Sciences RPN2232) reagent was used for detection and blots were imaged using ChemiDoc+ (Bio-Rad). Myosin heavy chain isoforms were separated as previously described [18 (link)], and gels were stained with SYPRO-Ruby (Bio-Rad 170-3138). Protein levels from all blots and gels were quantified using ImageJ.

Barefield D., Kumar M., Gorham J., Seidman J.G., Seidman C.E., de Tombe P.P, & Sadayappan S. (2014). Haploinsufficiency of MYBPC3 Exacerbates the Development of Hypertrophic Cardiomyopathy in Heterozygous Mice. Journal of molecular and cellular cardiology, 79, 234-243.

+ Open protocol

+ Expand

Fluorescent Visualization of Recombinant Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

A standard PURE reaction programmed with 17.5 nM (250 ng) of a linear Rep DNA template (under control of a T7 promoter) was supplemented with 0.6 µl FluoroTect™ Green_Lys tRNA (FluoroTect™ Green_Lysin vitro translation labelling system, Promega). Template DNA was omitted in the negative control reaction. Samples were incubated for 2 h at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific) and subsequently treated with 0.6 μl RNase Cocktail™ Enzyme Mix (0.5 U/μl RNase A and 20 U/μl RNase T1, Thermo Fisher Scientific) for 15 min at 37°C to degrade non-incorporated Green_Lys tRNA. 7.5 μl sample were mixed with an equal volume 2× Laemmli sample loading buffer (incl. 200 mM DTT) and denatured for 2.5 min at 65°C. Samples were analysed by conventional discontinuous SDS-PAGE (10% gel) run at 4°C (100 V for 10 min, then 200 V) on a Midi-format electrophoresis system (Atto). The fluorescent signal of the de novo expressed rep β subunit was imaged on a fluorescence laser scanner (Typhoon FLA 9000, GE Healthcare) at either 473 nm (blue LD laser/510LP filter) or at 532 nm (green SHG laser/575LP filter). Total protein and the molecular-weight size marker (PageRuler™ Unstained Protein Ladder, Thermo Fisher Scientific) were visualized after SYPRO Ruby (Bio-Rad) staining using the same instrument (473 nm, blue LD laser/575LP filter).

Weise L.I., Heymann M., Mayr V, & Mutschler H. (2019). Cell-free expression of RNA encoded genes using MS2 replicase. Nucleic Acids Research, 47(20), 10956-10967.

+ Open protocol

+ Expand

HMGB1 Protein Detection in Tissue Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tissue homogenates were lysed in RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6 at 4 °C) containing a cocktail of protease inhibitors (Calbiochem). Equal amounts of protein were separated by SDS-PAGE (4–12% Bis-Tris gels, Invitrogen) and transferred to PVDF membranes (Invitrogen), blocked in TBST plus 5% non-fat milk and then incubated with the following primary antibody: HMGB-1 (Abcam, ab18256) overnight at 4 °C. The membranes were then washed and TBST and incubated for 1 h at room temperature. The washed membranes were then treated with enhanced chemiluminescence detection reagent (Thermo Scientific). All Blots were developed using ChemiDoc XRS+ Molecular Imager (BioRad) and analyzed using Quantity One software (BioRad). Band densities were normalized to the total amount of protein loaded per lane using Sypro Ruby (BioRad).

Greco T., Vespa P.M, & Prins M.L. (2020). Alternative substrate metabolism depends on cerebral metabolic state following traumatic brain injury. Experimental neurology, 329, 113289.

+ Open protocol

+ Expand

In Vitro SUMO Conjugation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Reaction mixtures containing 20 mM HEPES (pH7.5), 50 mM NaCl, 10 mM MgCl₂, 0.1% Tween-20, 2 mM ATP, 1 mM DTT, 200 nM E1, 100 nM of indicated E2_Ubc9, 50 nM of the indicated Siz1^(167–465), 80 µM of the indicated Smt3, and 4 µM of the indicated PCNA were incubated at 30°C. Aliquots were removed at the indicated times and quenched in equal volume of 4X LDS NuPAGE loading dye with 1 M β-mercaptoethanol (β-me), resolved by 12% SDS-PAGE with MOPS running buffer. Proteins were stained with SYPRO Ruby (Bio-Rad) and imaged on Typhoon FLA 9500 with a 473-nm laser and an LPG filter. Band intensities were integrated with ImageJ and plotted against time in Excel to determine rates relative to wild type. Experiments were performed in triplicate.

Streich FC J.r, & Lima C.D. (2016). Capturing a substrate in an activated RING E3/E2-SUMO complex. Nature, 536(7616), 304-308.

+ Open protocol

+ Expand

UHRF1 Histone H3 Binding Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

40 μL streptavidin paramagnetic beads (MagneSphere, Promega) were washed three times with 1× PBS (pH 7.4) buffer and resuspended in 100 μL PD buffer (25 mM Tris, pH 7.5, 300 mM KCl, 20% glycerol, 1 mM DTT and 0.2% v/v Triton X-100). To pull down UHRF1, biotin-labeled H3_1-20K9me3 or unmodified H3_1-20, recombinant UHRF1 (residues 126-793) wild type or R649A/P656G mutant were mixed with the beads to a final concentration of 2.0 μM each and incubated at 4 °C for 1 hr. The beads were washed five times with PD buffer, followed by releasing the proteins into elution buffer (50 mM Tris, pH7.5, 25 % v/v glycerol, 1 mM EDTA, 2% SDS, 10 mM DTT) by boiling for 10 min. All the samples were finally resolved by SDS-PAGE and stained by Sypro ruby (Bio-rad).

Gao L., Tan X.F., Zhang S., Wu T., Zhang Z.M., Ai H.W, & Song J. (2018). An intramolecular interaction of UHRF1 reveals dual control for its histone association. Structure (London, England : 1993), 26(2), 304-311.e3.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!