Simplyblue safe stain solution

Manufactured by Thermo Fisher Scientific

Sourced in United States

SimplyBlue Safe stain solution is a Coomassie-based protein stain used for the visualization of proteins in polyacrylamide gels. It is a ready-to-use solution that provides a simple and safe staining procedure.

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SimplyBlue

13 protocols using simplyblue safe stain solution

Antibody-Based Detection of V. algivorus AlyB

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Affinity-purified rabbit polyclonal antibody recognizing a chemically synthesized peptide sequence of V. algivorus AlyB (AAQKEARKDLRK) (Eurofins Genomics, Tokyo, Japan) was prepared as previously described (Iwai et al. 2015 ). Rabbit polyclonal anti-AlyB antibody (1:400) and horseradish peroxidase-linked anti-rabbit IgG (3:2000; Cell Signaling Technologies, Danvers, MA, USA; catalogue no. 7074) were used to detect AlyB. Two independent repeats were carried out for western blot analyses, for which 2 μg of each sample was used. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out using XV Pantera pre-cast gels (DRC Co., Tokyo, Japan) and SimplyBlue SafeStain solution (Thermo Fisher Scientific).

Doi H., Tokura Y., Mori Y., Mori K., Asakura Y., Usuda Y., Fukuda H, & Chinen A. (2016). Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus. Applied Microbiology and Biotechnology, 101(4), 1581-1592.

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Confirming Recombinant Protein Size by SDS-PAGE

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In order to confirm recombinant proteins were correctly sized, SDS PAGE was performed using a BioRad gel electrophoresis system. Samples containing recombinant EK-PSA-GZMB/TRP proteins were mixed 1:1 with 4X reducing SDS PAGE Sample Buffer and boiled for 5 minutes. Samples were then loaded and run at 150V until fully migrated. For protein staining, the gel was then washed 2-3 times with RO H₂O and stained for one hour with SimplyBlue Safe Stain solution (Thermo LC6060). To de-stain samples were washed in RO H₂O overnight. For Western blotting, samples were run on a non-reducing gel and were transferred to PVDF Immuno-Blot membrane (BioRad 1620177) for 1 hour at 100V and incubated for one hour in TBST containing 5% milk. To monitor removal of the DDDK peptide, an anti-DDDDK polyclonal antibody (Abcam 1162) was diluted to 1:5000 in TBST/milk and incubated with the membrane overnight at 4°C. The next day, the membrane was washed 5X with TBST and incubated with an anti-rabbit HRP-linked IgG (Cell Signaling #70762) diluted at 1:10000 in TBST/milk for one hour at 4°C. The blot was then washed 5X with TBST and incubated with chemiluminescent substrate solution (Thermo 34077) diluted according to manufacturer's instructions. The blots were then developed at specified time points.

Rogers O.C., Anthony L., Rosen D.M., Brennen W.N, & Denmeade S.R. (2018). PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer. Oncotarget, 9(32), 22436-22450.

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Zymographic Analysis of Elastolytic MMP2

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Enzyme activity of elastolytic MMP2 was assayed using gel zymography, as described in prior studies by our group [27 (link)]. Briefly, cell layers (harvested in RIPA buffer containing protease inhibitor) were loaded into each lane of a 10% zymogram gel (Thermo Fisher Scientific). The gels were run for 2 h at 125 V in Tris-glycine SDS running buffer. Gels were then washed in a zymogram denaturing buffer (Thermo Fisher Scientific) for 30 min to remove any traces of SDS, and incubated in zymogram developing buffer (Thermo Fisher Scientific) overnight to activate the MMPs. Finally, the gels were stained (30 min) with a SimplyBlue^™ SafeStain solution (Thermo Fisher Scientific), and then distained in water, until clear bands were visibly apparent against the dark blue background of the gel. The intensities of the MMP2 bands in each case were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Fortin J.M., Hirota L.K., Bond B.E., O'Connor A.M, & Col N.F. (2001). Identifying patient preferences for communicating risk estimates: A descriptive pilot study. BMC Medical Informatics and Decision Making, 1, 2.

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SDS-PAGE Protein Analysis Protocol

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For SDS-PAGE analysis, supernatants from the harvest samples were treated with 4× Bolt^® LDS sample buffer (Life Technology, Frederick, MD, USA) which contains both lithium dodecyl sulfate as a denaturing agent and dithiothreitol (DTT) (Life Technology, Frederick, MD, USA) as a reducing agent. All samples were heated at 90 °C for 3 min before loading on SDS-PAGE gels. SDS-PAGE was performed using 10% pre-cast Bis–Tris NuPAGE SDS gels (Life Technology, Frederick, MD, USA). Electrophoresis was performed at a constant 200 V for 45 min in MOPS running buffer under denaturing conditions (Life Technology, Frederick, MD, USA). The separated protein bands were visualized by staining with the Simply Blue Safe Stain solution (Life Technology, Frederick, MD, USA) or transferred onto nitrocellulose membranes for Western blot analysis.

Han S., Machhi S., Berge M., Xi G., Linke T, & Schoner R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express, 7, 93.

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Gelatin Zymography for MMP9 Activity

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Cell-conditioned medium was analyzed for MMP9 activity by Gelatin zymography. Cells were incubated in serum-free media for 24 h and then the conditioned medium was collected and concentrated. Samples were prepared in non-reducing conditions in 5× sample buffer (0.625 M Tris-HCl, 10% glycerol, 2% SDS, 2% Bromphenol Blue) and resolved on 8% SDS-PAGE gels containing 0.5 mg/ml of gelatin Type A from porcine skin (Sigma-Aldrich). The gels were then washed twice in 2.5% (v/v) Triton X-100 for 15 min each at room temperature. Following this, gels were incubated in developing buffer (100 mM Tris-HCl, pH 7.9, 30 mM CaCl₂, and 0.02% sodium azide) for 30 min at room temperature followed by a second incubation in fresh developing buffer overnight at 37°C. The gels were then stained with SimplyBlue SafeStain solution (Life Technologies) for 15 min, followed by de-staining in water to reveal areas of activity [91 (link)].

Baldwin R.M., Haghandish N., Daneshmand M., Amin S., Paris G., Falls T.J., Bell J.C., Islam S, & Côté J. (2014). Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression. Oncotarget, 6(5), 3013-3032.

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Characterization of PARP-1-DNA Complexes

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After intrinsic cross-linking of polypeptides I and II with ³²P-labeled AP site-containing DNA as above, the pH of the of reaction mixture was adjusted to 8.0 by adding 1 M Tris-HCl, pH 8.0, to a final concentration of 100 mM. An aliquot was withdrawn before adding trypsin to the reaction mixture containing the PARP-1 and DNA complex. To the remaining reaction mixture, trypsin was added at a 1:5 weight ratio of trypsin to polypeptide, and the mixture was incubated for 20 min at 25°C. The reaction was terminated by adding 100 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, SDS-PAGE sample buffer, and boiling for 5 min. The digested products were separated by Nu-PAGE Bis-Tris gel (4–12%), and a PhosphorImager was used to detect radiolabeled cross-linked peptides. Then, the same gel was stained for proteins detection with SimplyBlue SafeStain solution (Life Technologies).

Prasad R., Horton J.K., Chastain PD I.I., Gassman N.R., Freudenthal B.D., Hou E.W, & Wilson S.H. (2014). Suicidal cross-linking of PARP-1 to AP site intermediates in cells undergoing base excision repair. Nucleic Acids Research, 42(10), 6337-6351.

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Detailed Hair Keratin Extraction Protocol

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Hydrogen peroxide (H2O2), sodium sulphide (Na2S), N-Ethylmaleimide (NEM) and dialysis tubings (molecular weight cut-off 12,400 Da) were purchased from Sigma Aldrich. Dulbecco's modified Eagle's medium (DMEM), L-glutamine, sodium pyruvate, 0.25% trypsin, PicoGreen DNA quantification kit, antibody-antimycotic mixture, secondary goat anti-mouse horseradish-peroxidase conjugated (HRP) antibody, CM-H2DCFDA, SimplyBlue SafeStain solution, precast 4-12% Bis-TRIS NuPAGE gels, lithium dodecyl sulphate sample buffer, sample reducing agent, MOPS SDS running buffer, NuPAGE antioxidant, and iBlot gel transfer stacks were purchased from Life Technologies. Primary mouse polyclonal antibody against total human hair keratins (clone AE13, #ab16113) was obtained from Abcam. The Super Signal West Pico chemiluminescent substrate was purchased from Pierce.

Lai H.Y., Wang S., Singh V., Nguyen L.T.H, & Ng K.W. (2018). Evaluating the antioxidant effects of human hair protein extracts. Journal of biomaterials science. Polymer edition, 29(7-9).

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SDS-PAGE and Western Blot Analysis

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Protein fractions were also analyzed by SDS-PAGE denaturing gels followed by staining with SimplyBlue SafeStain solution (Invitrogen) or Western blotting as described (15 (link)). For Western blot analysis, the following primary antibodies were used: BRCA1 (C-20; Santa Cruz Biotechnology, sc-642), ubiquitin-pAb (Enzo Life Sciences, ADI-SPA-200), p53 (DO-1; Santa Cruz Biotechnology, sc-126), K63-linkage specific polyubiquitin (D7A11; Cell Signaling, #5621) and β-actin (Sigma-Aldrich, A5441). Western blot quantification and densitometry measurements were performed using Image Lab™ Software (Bio-Rad). The band intensities were selected using the volume tool. Local subtraction and linear regression methods were implemented to eliminate the local background values and to quantify the band intensities.

Liang Y., Dearnaley W.J., Alden N.A., Solares M.J., Gilmore B.L., Pridham K.J., Varano A.C., Sheng Z., Alli E, & Kelly D.F. (2018). Correcting Errors in the BRCA1 Warning System. DNA repair, 73, 120-128.

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Characterization of Protein Uptake Mechanisms

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Gold chloride trihydrate (HAuCl₄·3H₂O, G4022), FITC–dextran (20 kDa, ~3.3 nm, FD20S), cell culture-grade dimethyl sulfoxide (DMSO, D4540) and d-glucosamine (2DG, G4875) were purchased from Sigma-Aldrich. High-purity bovine α-LA (Bos taurus) was provided by Agropur (Ca²⁺ < 0.055%). The purity was further verified by SDS–PAGE on a Novex NuPAGE (NP0335PK2, Invitrogen) 4−12% Bis-Tris precast protein gel using SimplyBlue SafeStain solution (LC6065, Invitrogen) for Coomassie Brilliant Blue G-250 staining. EIPA (3378) and cytochalasin D (1233) were obtained from Tocris Bioscience. NVP-BEZ235 (SYN-1018), wortmannin (AC32859) and staurosporine (ALX-380-014) were bought from AdipoGen, Acros Organics and Enzo Life Sciences, respectively. Sulfo-Cy5.5 NHS ester was purchased from Lumiprobe. Methanol-free paraformaldehyde (PFA; 16%, 0219998380) was bought from MP Biomedicals. All of the chemicals were analytical grade unless otherwise noted. Ultrapure 18.2 MΩ-cm Milli-Q water was used throughout the study. All PBS used in the study was 1× and diluted from 10× stock (BP3994, Thermo Fisher Scientific) as Ca²⁺ and Mg²⁺ free 11.9 mM pH 7.4 with 137 mM NaCl and 2.7 mM KCl, unless otherwise specified.

Yang J., Wang T., Zhao L., Rajasekhar V.K., Joshi S., Andreou C., Pal S., Hsu H.T., Zhang H., Cohen I.J., Huang R., Hendrickson R.C., Miele M.M., Pei W., Brendel M.B., Healey J.H., Chiosis G, & Kircher M.F. (2020). Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer. Nature biomedical engineering, 4(7), 686-703.

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SDS-PAGE Analysis of Recombinant Proteins

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Recombinant proteins or bovine serum albumin (1 µg each) were mixed with SDS sample buffer (Invitrogen, CA, USA) and subjected to SDS-PAGE. The gel was treated with SimplyBlue Safe stain solution (Invitrogen, CA, USA) and incubated until blue bands appeared on the gel [10 (link),11 (link)].

Kato K, & Tachibana H. (2022). Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities. International Journal of Molecular Sciences, 23(14), 7700.

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