Instantblue coomassie

Manufactured by Abcam

Sourced in United States, United Kingdom

InstantBlue Coomassie is a ready-to-use Coomassie protein stain for the rapid detection of proteins in polyacrylamide gels. It provides a simple, fast, and sensitive method for protein visualization.

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Lab products found in correlation

Typhoon trio scanner, by GE Healthcare (3 mentions) Penta his antibody, by Qiagen (3 mentions) Alexa fluor 647, by Thermo Fisher Scientific (3 mentions) Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by GE Healthcare (2 mentions) Tris acetate sds running buffer, by Thermo Fisher Scientific (1 mentions) Iblottm 2 dry blotting system, by Thermo Fisher Scientific (1 mentions) 1 steptm nbt bcip, by Thermo Fisher Scientific (1 mentions) Nativepage 3 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Gradient gel, by GenScript (1 mentions) Odyssey, by LI COR (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) C1000 thermal cycler, by Bio-Rad (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Image lab 3, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Expicho, by Thermo Fisher Scientific (1 mentions)

19 protocols using instantblue coomassie

Native PAGE and Western Blot Analysis

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Samples were mixed with NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific) and were analysed on NativePAGE 3–12% Bis-Tris Gel (Thermo Fisher Scientific). The gels were run at 50 V for 20 min and 100 V for an additional 2 h in the Tris-acetate SDS running buffer (Thermo Fisher Scientific). For western blot, samples were mixed with LDS Sample Buffer and reducing agent, boiled, and loaded on NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific). The gels were stained with the InstantBlue Coomassie (Abcam) for 10 min before analysing with ChemiDoc™ Touch Imaging System (Bio-Rad). J chain western blots were performed using iBlot^TM 2 Dry Blotting System (Thermo Fisher Scientific) and iBind^TM Flex western system (Thermo Fisher Scientific) following the manufacturer’s protocol. Antibodies: Rabbit anti-J Chain (SP105, 1:100, Thermo Fisher Scientific), Goat anti-Rabbit IgG1, AP conjugated (1:1,000, Thermo Fisher Scientific), and 1-Step^TM NBT/BCIP (Thermo Fisher Scientific) were used for detection.

Guo H., Cho B., Hinton P.R., He S., Yu Y., Ramesh A.K., Sivaccumar J.P., Ku Z., Campo K., Holland S., Sachdeva S., Mensch C., Dawod M., Whitaker A., Eisenhauer P., Falcone A., Honce R., Botten J.W., Carroll S.F., Keyt B.A., Womack A.W., Strohl W.R., Xu K., Zhang N., An Z., Ha S., Shiver J.W, & Fu T.M. (2023). An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV. Emerging Microbes & Infections, 12(2), 2275598.

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Oligomeric State Analysis of BbmrR

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The oligomeric state of BbmrR was determined via analytical size-exclusion chromatography on a Superdex 200 10/300 gel ltration column (Cytiva). The column was pre-equilibrated with 50 mM phosphate buffer, 500 mM NaCl, pH 7.5, and 100 mL of protein sample were injected. For calibration of the column, a series of proteins from the standard gel-ltration calibration LMW/HMW kits (Cytiva) were used. SDS-PAGE samples were prepared by mixing a concentration of 5-20 mg protein with standard Coomassie loading dye and incubated at 95 °C for 10 min. Samples were centrifuged (except for CE) and loaded into a precasted 4-20% gradient gel (GenScript) for 1 h at 135 V and stained using Instant Blue Coomassie (Abcam).

Gran-Scheuch A., Hanreich S., Keizer I., W Harteveld J., Ruijter E, & Drienovská I. (2024). Designing Michaelases: exploration of novel protein scaffolds for iminium biocatalysis. Faraday discussions.

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Liposome-based PGRN Binding Assay

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Liposomes were prepared in HBSS (1mg/ml final lipid concentration) as described above. 200μL of these liposome preparations were spun for 30 min at 100,000 x g at 22°C and supernatants were carefully removed. Liposome pellets were resuspended in 75μL of either MES buffer pH 4.5 (30mM MES, 110mM KCl, 20mM NaCl) or HEPES buffer pH 7.0 (30mM HEPES, 110mM KCl, 20mM NaCl). Liposomes were incubated for 20 min at room temperature with 4μg of recombinant human PGRN protein (Cat # 2420-PG, R&D systems, USA) on a horizontal shaker. Samples were then centrifuged at 100,000 x g for 30 min at 22°C and 75μL of supernatants were harvested. Liposome pellets were resuspended in 100μL of 1X LDS buffer (containing 4X LDS, Cat # NP007, Invitrogen; 10X Reducing agent, Cat # NP0009, Invitrogen; Protease Inhibitors, Roche and PBS) and supernatants were resuspended in 25μL of 4X LDS buffer (containing 4X LDS, Cat # NP007, Invitrogen; 10X Reducing agent, Cat # NP0009, Invitrogen; Protease Inhibitors, Roche). Samples were boiled for 5 min at 95°C and run on an SDS-PAGE, prior to staining of the gel with Coomassie Blue (InstantBlue® Coomassie, Abcam # 119211, USA). Image acquisition was performed with the 700nm channel on a LI-COR Odyssey (LI-Cor Biosciences) and protein signals were quantified using Image J.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

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SDS-PAGE Analysis of Phytase and β-Lactamase Constructs

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SDS-PAGE was run on 12% (for phytase constructs and heat purification gels) or 14% (for β-lactamase constructs) polyacrylamide gels in an XCell SureLock module (Life Technologies). Gels were run at 200 V for 1 hour and then stained with InstantBlue Coomassie (Expedeon). For β-lactamase constructs, a final concentration of 100 mM dithiothreitol (DTT, Sigma) was added. For phytase constructs, a final concentration of 100 mM ethylenediaminetetraacetic acid (EDTA) was added to remove tightly-bound divalent cations affecting phytase mobility. Samples were mixed with 6× SDS-PAGE loading buffer (0.23 M Tris-HCl, 0.24% glycerol, 6.7% SDS, and 12 mM bromophenol blue) and heated at 95 °C for 7 minutes in a Bio-Rad C1000 Thermal cycler. Gels were imaged on a Bio-Rad ChemiDoc XRS+ and analyzed using Image Lab 3.0 software (Bio-Rad).

Schoene C., Bennett S.P, & Howarth M. (2016). SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries. Scientific Reports, 6, 21151.

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Rab8a Dephosphorylation Assay by PPM1H

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In vitro dephosphorylation assay was performed in a total volume of 20 µl in 40 mM Hepes buffer (pH 7.0) containing 10 mM MgCl₂ using 2.5 µg pT72 phosphorylated Rab8a[Q67L] and varying levels of recombinant PPM1H. 3 µl of pT72 phosphorylated Rab8a[Q67L] at 0.83 mg/ml in a buffer containing 20 mM MES pH 5.3, 0.1 M NaCl, 10% (by vol) glycerol, 0.03% (by vol) Brij 35, 14 mM 2-mercaptoethanol, 2 mM MgCl₂, 1 µM GTP-γ-S added to the assay. The assay was initiated by addition of 2 µl of serial dilutions of PPM1H (0.1 mg/mL, 0.02 mg/mL, 0.004 mg/mL, 0.0008 mg/mL) diluted into HEPES Buffer from a stock of 1.73 mg/ml PPM1H in 50 mM Tris/HCl pH7.5, 150 mM NaCl, 2 mM MnCl2, 0.5 mM TCEP Buffer. The assay was carried out for 30 min and terminated by addition of 6 µl 4 x LDS (106 mM Tris HCl, 141 mM Tris Base, 2% (by mass) LDS, 10% (by vol) glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5) with 5% (by vol) 2‐mercaptoethanol. Samples were then subjected to Phos-tag gel electrophoresis to determine stoichiometry of phosphorylated Rab8a as described previously in Ito et al. (2016) (link). Gel was stained using Instant Blue Coomassie (Expedeon).

Berndsen K., Lis P., Yeshaw W.M., Wawro P.S., Nirujogi R.S., Wightman M., Macartney T., Dorward M., Knebel A., Tonelli F., Pfeffer S.R, & Alessi D.R. (2019). PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins. eLife, 8, e50416.

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Nanobodies Purification and Validation

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CD4-specific Nbs were expressed and purified as previously published (71 (link), 75 (link)). Extracellular fragment of hCD4 comprising domains 1–4 of hCD4 and a C-terminal His6-tag was expressed in Expi293 cells according to the manufacturer’s protocol (TFS). Cell supernatant was harvested by centrifugation 4 days after transfection, sterile filtered and purified according to previously described protocols (76 (link)). For quality control, all purified proteins were analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures. Therefore, protein samples were denaturized (5 min, 95°C) in 2× SDS sample buffer containing 60 mM Tris/HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% bromphenole blue. All proteins were visualized by InstantBlue Coomassie (Expedeon) staining. For immunoblotting, proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and detection was performed using anti-His primary antibody (Penta-His Antibody, #34660, Qiagen) followed by donkey anti-mouse secondary antibody labeled with Alexa Fluor 647 (Invitrogen) using a Typhoon Trio scanner (GE Healthcare, excitation 633 nm, emission filter settings 670 nm BP 30).

Traenkle B., Kaiser P.D., Pezzana S., Richardson J., Gramlich M., Wagner T.R., Seyfried D., Weldle M., Holz S., Parfyonova Y., Nueske S., Scholz A.M., Zeck A., Jakobi M., Schneiderhan-Marra N., Schaller M., Maurer A., Gouttefangeas C., Kneilling M., Pichler B.J., Sonanini D, & Rothbauer U. (2021). Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells. Frontiers in Immunology, 12, 799910.

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SDS-PAGE Protein Quantification

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A total of 3 μg of each protein was prepared in SDS–PAGE sample buffer and 5% 2‐mercaptoethanol. Electrophoresis was carried out using NUPAGE Bis–Tris 4–12% gradient gels (Life Technologies) and run at 120 V. The gel was then stained for 1h using Instant Blue Coomassie (Expedeon). Protein concentrations were adjusted using the upper protein band, which corresponded to the undegraded protein.

Waschbüsch D., Berndsen K., Lis P., Knebel A., Lam Y.P., Alessi D.R, & Khan A.R. (2021). Structural basis for the specificity of PPM1H phosphatase for Rab GTPases. EMBO Reports, 22(11), e52675.

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Nanobody Expression and Purification

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Miro1-Nbs were expressed and purified as previously described (Maier et al., 2015 (link); Wagner and Rothbauer, 2021 (link)). Bivalent Nbs were expressed using the ExpiCHO™ system (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. For quality control, all purified proteins were analyzed by SDS-PAGE according to standard procedures. Protein samples were denatured (5 min, 95°C) in 2x SDS-sample buffer containing 60 mM Tris/HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.02% bromphenol blue prior to analysis. All proteins were visualized by InstantBlue Coomassie (Expedeon) staining. For immunoblotting, proteins were transferred to nitrocellulose membrane (GE Healthcare) and detection was performed using anti-His primary antibody (Penta-His Antibody, #34660, Qiagen) followed by donkey-anti-mouse secondary antibody labelled with AlexaFluor647 (Invitrogen). A Typhoon Trio scanner (GE-Healthcare, excitation 633 nm, emission filter settings 670 nm BP 30) was used for the readout of fluorescence signals.

Fagbadebo F.O., Kaiser P.D., Zittlau K., Bartlick N., Wagner T.R., Froehlich T., Jarjour G., Nueske S., Scholz A., Traenkle B., Macek B, & Rothbauer U. (2022). A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1. Frontiers in Molecular Biosciences, 9, 835302.

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Analytical Methods for Shell Extracts

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The shell extracts were analysed by conventional mono-dimensional SDS-PAGE (Bio-Rad, Mini Protean III gels), on precast gradient gels (Mini-PROTEAN TGX Gel 4–20% acrylamide, 90 mm x 70 mm, BioRad). Prior migration, the samples were treated as follows: lyophilisates of the ASM were dissolved in 2X Laemmli Sample Buffer (LSB) to a final matrix concentration of 5 μg/μL. One lyophilisate of the AIM was suspended in 2X LSB (5 mg in 500 μL). Both preparations were heat-denatured at 100°C: 5 min. for the ASM and 10 min. for the AIM. The LSB-solubilized fraction of the AIM is referred as LS-AIM (Laemmli soluble, acetic acid insoluble). The preparations were cooled down and gently centrifuged before being applied on the top of the gel: 20 μg of ASM and 50 μg of LS-AIM were applied, respectively. After migration, the gels were stained with Instant Blue Coomassie (Expedeon, Harston, UK), with silver nitrate [52 (link)], with Alcian Blue at low pH (pH 1) [33 (link)–53 (link)] and with ‘Stains-all’ [54 (link)]. While silver nitrate is supposed to stain most of the macromolecular components of the shell, Alcian Blue in acidic conditions stains mostly sulphate groups of polyanionic polysaccharides. ‘Stains-all’ stains blue the putative calcium-binding proteins, while leaving the non-calcium-binding proteins red or pink.

Immel F., Broussard C., Catherinet B., Plasseraud L., Alcaraz G., Bundeleva I, & Marin F. (2016). The Shell of the Invasive Bivalve Species Dreissena polymorpha: Biochemical, Elemental and Textural Investigations. PLoS ONE, 11(5), e0154264.

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Native Protein Gel Electrophoresis

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Novex^™ WedgeWell^™ 4–12% Tris-glycine gels were used. The gel chamber (Invitrogen) was cooled in an ice bath, and the gel electrophoresis was conducted in a cold room (4 °C) using NativePAGE running buffer (Invitrogen). The gel electrophoresis was carried out for 1.5 h at 160 V, and the gel was stained using InstantBlue Coomassie (Expedeon).

Reinbold R., Hvinden I.C., Rabe P., Herold R.A., Finch A., Wood J., Morgan M., Staudt M., Clifton I.J., Armstrong F.A., McCullagh J.S., Redmond J., Bardella C., Abboud M.I, & Schofield C.J. (2022). Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors. Nature Communications, 13, 4785.

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