Simplyblue

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

SimplyBlue is a protein staining solution used for visualizing proteins in polyacrylamide gels. It is a simple and effective method for detecting proteins separated by gel electrophoresis.

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Novex® SimplyBlue™ SafeStain SimplyBlue SafeStain staining solution Coomassie SimplyBlue SafeStain SimplyBlue Coomassie stain SimplyBlue staining SimplyBlue SafeStain SimplyBlue™ SafeStain kit Coomassie G-250 (SimplyBlue Safestain™; SimplyBlue Safe stain solution SimplyBlue Coomassie

65 protocols using simplyblue

Protein Aggregation Analysis via SDS-PAGE

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Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE Novex bis-Tris 12-well precast gels (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA), containing 4–12% polyacrylamide, prepared according to the manufacturer’s instructions under reducing and non-reducing conditions in order to study the possible aggregation of proteins using MES (2-(N-Morpholino) ethanesulfonic acid hemisodium salt) buffer. Sample volumes (10 µL) were injected into each well to achieve a protein loading of 0.65 mg/mL. A fixing solution (50% methanol and 10% acetic acid in v/v) was applied to the gels for 2 h before staining with the commercial staining solution SimplyBlue^TM (Thermo Scientific, Vilnius, Lithuania). Thermo Scientific’s PageRuler Unstained Low Range Protein Ladder, with its mixture of purified proteins and peptides in the size range 3.4 to 100 kDa, was used as molecular size markers.

Sáez L., Murphy E., FitzGerald R.J, & Kelly P. (2019). Exploring the Use of a Modified High-Temperature, Short-Time Continuous Heat Exchanger with Extended Holding Time (HTST-EHT) for Thermal Inactivation of Trypsin Following Selective Enzymatic Hydrolysis of the β-Lactoglobulin Fraction in Whey Protein Isolate. Foods, 8(9), 367.

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Zymogram Gelatin Protein Visualization

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Precast 10% zymogram (gelatin) protein gels (Novex TM , ThermoFisher Scientific)
were loaded with 20 μL of tissue culture medium, pre-spun at 1200 rpm for 5 min to remove cellular debris. Following SDS-PAGE, gels were renatured and developed in the respective buffers (Novex TM ) and finally developed for 24 hours at 37°C. Gels were then stained with SimplyBlue TM (ThermoFisher Scientific) and scanned.

Kalafatovic D., Nobis M., Son J., Anderson K.I, & Ulijn R.V. (2016). MMP-9 triggered self-assembly of doxorubicin nanofiber depots halts tumor growth. Biomaterials, 98.

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SDS-PAGE Analysis of Helleramine Fraction

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A total of 20 µg of fraction containing helleramine was run on a NuPAGE® Novex 4–12% (w/v) Bis-Tris SDS-PAGE gels (Invitrogen™, Carlsbad, CA, USA). An XCell SureLock™ at 150 V for 90 min using a SureXCell system (Invitrogen, CA, USA). The gel was stained with SimplyBlue (Invitrogen, CA, USA) for 1 h and distained overnight with 18-megaOhm water. SeeBlue Plus2 markers, ranging from 3–188 kDa, were used as standards.

Salazar E., Rodriguez-Acosta A., Lucena S., Gonzalez R., McLarty M.C., Sanchez O., Suntravat M., Garcia E., Finol H.J., Giron M.E., Fernandez I., Deba F., Bessac B.F, & Sánchez E.E. (2020). Biological activities of a new crotamine-like peptide from Crotalus oreganus helleri on C2C12 and CHO cell lines, and ultrastructural changes on motor endplate and striated muscle. Toxicon : official journal of the International Society on Toxinology, 188, 95-107.

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In Vitro Phosphorylation of LCN2 by PKCδ

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In vitro phosphorylation of purified LCN2 by recombinant PKCδ (Invitrogen) was performed as described [22 (link)]. The kinase reaction was initiated at 37°C by adding recombinant LCN2 and [γ-³²P]ATP. At different time points, the reaction mixture was stopped by the addition of SDS sample buffer. Proteins were separated on NuPAGE Bis-Tris gels (Invitrogen) and stained using SimplyBlue (Invitrogen). Phosphorylated LCN2 was detected by phosphorimaging (Typhoon 9410, Amersham Bioscience).

Weng Y.C., Wang G., Messing R.O, & Chou W.H. (2015). Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils. Journal of Biomedical Science, 22(1), 21.

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Quantitative Protein Analysis in C2C12 Myotubes

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We homogenized C2C12 myotubes in 2X protein loading buffer (120 mM Tris pH 7.5, 4% SDS, 200 mM DTT, 20% glycerol, 0.002% bromphenol blue). Proteins were separated in equal volumes of lysates by SDS-PAGE (4-15% Criterion, BioRad). We determined relative total protein by scanning (Odyssey Infrared Imaging, LI-COR) stained gels (Simply Blue, Invitrogen). We used fluorescence intensity data to normalize total protein for equal loading. SDS-PAGE was used to separate equal amounts of protein, which was then transferred to PVDF membranes for western blot using the Odyssey System (Moylan et al., 2014 (link)). For conditioned media samples, we combined 6X loading buffer (as above) 1:10 with conditioned media, and followed the same procedure as for lysates.

McLean J.B., Moylan J.S., Horrell E.M, & Andrade F.H. (2015). Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia. Frontiers in Physiology, 6, 136.

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Gelatin Zymography for MMP-2 Activity

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Polyacrylamide/sodium dodecyl sulfate (SDS) gel electrophoresis (Page) was performed using 10% gels containing 1 mg/mL of gelatin (Sigma, Germany). SDS was removed by washing in 2.5% Triton X-100 for 1 h at room temperature before the enzyme reaction. The gel was incubated overnight at 37 °C in enzyme buffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl2, and 0.02% Brij-35. The MMP-2 activity was identified by the area where the gelatin was degraded which appears as a distinct white band after staining the gel with simply blue (Invitrogen, Germany) for 2 h at room temperature followed by overnight washing with water. The imaging and analysis were done using the same methodology as for western blot analysis.

Sarem M., Otto O., Tanaka S, & Shastri V.P. (2019). Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis. Stem Cell Research & Therapy, 10, 10.

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Gelatin Zymography for MMP-9 Analysis

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Electrophoresis was performed with 10% polyacrylamide/sodium dodecyl sulfate gels containing 1 mg/ml of gelatin (Sigma, Germany). Sodium dodecyl sulfate was removed by washing in 2.5% Triton X-100 for 1 h at room temperature before the enzyme reaction. The gel was incubated overnight at 37°C in enzyme buffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl₂, and 0.02% Brij-35. Area of gelatin degradation, identified as MMP activity, appeared as distinct white band after staining the gel with simply blue (Invitrogen, Germany). The intensities of both pro- and active MMP-9 were documented by a digital gel-imaging system (Thermo Scientific) and bands were analyzed by densitometry using imageJ.

Christensen J, & Shastri V.P. (2015). Matrix-metalloproteinase-9 is cleaved and activated by Cathepsin K. BMC Research Notes, 8, 322.

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Evaluating Kif15-Tpx2 Interactions

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Glycerol gradients were essentially carried out as described in McClelland and McAinsh (2009) (link) in the presence of 1 mM DTT and complete protease protein inhibitor at 4°C for 15 hr over night. We checked for hKif15/hTpx2 interactions in 50 mM HEPES pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM ATP, 0/150/300/450 mM NaCl (150 mM NaCl also ± free tubulin in ATP or AMP-PNP) and 35 mM sodium phosphate buffer pH7.0, 1 mM MgCl₂, 1 mM EGTA, 0/150/300/500 mM NaCl, 0.1 mM ATP or AMP-PNP. The 5-mlgradient was fractionated by hand in fractions of 200 µl each. The protein was TCA-precipitated and fractions 1–23, together with ¹/₂ input, analysed on the same SDS-PAGE gel stained with coomassie (SimplyBlue, Invitrogen). Protein bands were quantified with ImageJ and peak values were determined by fitting a Gaussian distribution to the data using Origin 8.5. Selected proteins from the High/low molecular weight gel filtration calibration kit (GE Healthcare) were used in standard calibration runs.

Drechsler H., McHugh T., Singleton M.R., Carter N.J, & McAinsh A.D. (2014). The Kinesin-12 Kif15 is a processive track-switching tetramer. eLife, 3, e01724.

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Venom Protein Separation and Identification

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A total of 25 µg of crude venom of each individual was separated on a non-reduced 4–20% Tricine Gel for 90 min at a voltage of 125 using an XCell SureLock® Mini-Cell system(Invitrogen). Current was moderated using a Bio-Rad PowerPack power supply. Protein was transferred onto a 0.2 µm nitrocellulose membrane (Millipore) using a Trans Blot SD system (Bio-Rad) at 100 mA for 1 h and allowed to set overnight. Duplicate gels under the same conditions were stained with Simply Blue (Invitrogen) for 1 h. The membrane was then blocked with 5% BSA in TBST for 1 h, washed with PBS and incubated with the anti-r-mojastin 1 antibody (1:1000 dilution) overnight at room temperature. The membrane was washed three times with TBS and incubated with a biotinylated goat anti-rabbit antibody (1:30,000 dilution) for 1 h at room temperature. The membrane was washed three times with 0.05% Tween 20 in PBS and incubated with ExtrAvidin-peroxidase (SIGMA, USA) diluted to 1:2000 in PBS for 1 h at room temperature. After washing off the unbound avidin with TBST, the antigen-bound antibody was visualized with SigmaFAST™ 3,3′-diaminobenzidine tablets. Bands of interest were transferred to nitrocellulose using the same conditions, as previously described, excised, and sent for N-terminal sequencing at the Iowa State Sequencing Facility.

Cantú E J.r., Mallela S., Nyguen M., Báez R., Parra V., Johnson R., Wilson K., Suntravat M., Lucena S., Rodríguez-Acosta A, & Sánchez E.E. (2016). The binding effectiveness of anti-r-disintegrin polyclonal antibodies against disintegrins and PII and PIII metalloproteases: An immunological survey of type A, B and A + B venoms from Mohave rattlesnakes. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 191, 168-176.

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Quantification and Purification of SARS-CoV-2 Spike Protein

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Approximately 2440 μg of total viral protein from HCoV-NL63 particles was resolved by SDS-PAGE gel (Bio-Rad) and stained with SimplyBlue^™ (Invitrogen). The amount of S protein was estimated to be 10% of the total protein based on the intensity of the stained bands. Bands at 250 kDa (S protein) were cut and then combined to make 10-μg aliquots of S protein.

Chmielewski D., Wilson E.A., Pintilie G., Zhao P., Chen M., Schmid M.F., Simmons G., Wells L., Jin J., Singharoy A, & Chiu W. (2023). Integrated analyses reveal a hinge glycan regulates coronavirus spike tilting and virus infectivity. Research Square.

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