Silverquest staining kit

Manufactured by Thermo Fisher Scientific

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The SilverQuest staining kit is a tool used to visualize proteins in polyacrylamide gels. It provides a sensitive and reliable method for protein detection and quantification.

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57 protocols using silverquest staining kit

Purification and Characterization of Anti-V2 Fab Fragments

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F(ab′)2 fragments were prepared from both NCI05 or NCI09 mAb, as these antibodies recognize overlapping conformationally distinct V2 epitopes^{16 (link)}, using Pierce F(ab′)2 Micro Preparation Kit (cat. no. 44688, Thermo Fisher) following the manufacturer’s instructions. An SDS–PAGE gel with the recovered F(ab′)2 was run and Silver stained (cat. no. LC6070, Silver Quest staining Kit, Invitrogen) according to the manufacturer’s instructions, to assure the purity of the F(ab′)2 fragments. Target cells, coated with ΔV1 gp120 protein as indicated above and labelled with SNAP-Surface Alexa Fluor 647, were incubated for 1 h at 37 °C with 5 μg ml⁻¹ of purified F(ab′)2 fragments from NCI05, or NCI09 monoclonal antibodies. Cells incubated without F(ab′)2 served as control. These target cells were subsequently used in the ADCC assay as described above. These F(ab′)2 inhibit binding (and ADCC) mediated by the anti-V2 antibodies from immunized animals’ plasma. The percentage ADCC activity difference in the presence or absence of F(ab′)2 is considered V2-specific ADCC activity.

Rahman M.A., Bissa M., Silva de Castro I., Helmold Hait S., Stamos J.D., Bhuyan F., Hunegnaw R., Sarkis S., Gutowska A., Doster M.N., Moles R., Hoang T., Miller Jenkins L.M., Appella E., Venzon D.J., Choo-Wosoba H., Cardozo T., Baum M.M., Appella D.H., Robert-Guroff M, & Franchini G. (2023). Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques. Nature Microbiology, 8(5), 905-918.

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Adsorption of Protein Antigens and CpG onto Alum Adjuvant

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The vaccines (50 μL/dose/mouse) were prepared on the day of vaccination. As previously described [19 (link), 26 (link), 29 (link)], Alhydrogel (100 μg/dose per mouse based on aluminum ion) was incubated in histidine buffer with or without phosphate buffer at 5 °C for 1 hr. Proteins (A33V, B5V, L1V, A27V) in histidine buffer were then added at 2 μg/dose or 10 μg/dose per mouse. CpG (50 μg/dose) was then added and formulations with or without CpG were incubated at 5 °C for an additional ~5 h. The adsorption status of proteins (and CpG) to AH and PTAH were determined by SDS-PAGE (4–15% gradient Mini-Protean TGX gel) under reducing and denaturing conditions. Protein bands and CpG bands were visualized in the gels by silver staining (Invitrogen Silverquest Staining Kit) following the manufacturer’s instructions.

Xiao Y., Zeng Y., Schante C., Joshi S.B., Buchman G.W., Volkin D.B., Middaugh C.R, & Isaacs S.N. (2020). Short-term and longer-term protective immune responses generated by subunit vaccination with smallpox A33, B5, L1 or A27 proteins adjuvanted with aluminum hydroxide and CpG in mice challenged with vaccinia virus. Vaccine, 38(38), 6007-6018.

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Purification of PRDM9 Complex

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The PRDM9 complex was purified by immunoprecipitation (IP) using anti-FLAG (IP-FLAG) and -HA antibodies (IP-HA). About 35 mg of proteins from each nuclear fraction were used. FLAG affinity purification was performed with EZview anti-FLAG M2 Affinity Gel (Sigma). Elution was performed with 0.2 mg/ml of FLAG peptide. HA affinity purification was performed with anti-HA beads (Santa Cruz). Elution was performed with 0.4 mg/ml HA peptide (eluate 1 and 2) and 2 mg/ml HA peptide (eluate 3). Eluates 1 and 2 were analyzed on 4–15% acrylamide gels by silver staining (Silver Quest Staining Kit, Invitrogen).

Imai Y., Biot M., Clément J.A., Teragaki M., Urbach S., Robert T., Baudat F., Grey C, & de Massy B. (2020). PRDM9 activity depends on HELLS and promotes local 5-hydroxymethylcytosine enrichment. eLife, 9, e57117.

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Preparation and Characterization of V2-Specific F(ab')2 Fragments for ADCC Assay

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F(ab’)2 fragments were prepared from NCI05 or NCI09 mAb, as these antibodies recognize overlapping conformationally distinct V2 epitopes (27 (link)), using Pierce F(ab’)₂ Micro Preparation Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. SDS-page gel with the recovered F(ab’)₂ was run and silver stained (Silver Quest staining Kit, Invitrogen) according to the manufacturer’s instructions, to assure the purity of the F(ab’)2 fragments. Target cells, coated with gp120 as indicated and labeled with SNAP-Surface^® Alexa Fluor^® 647, were incubated for 1 h at 37°C with 5 μg/ml of purified F(ab’)₂ fragments from NCI05 or NCI09 monoclonal antibodies. Cells incubated without F(ab’)₂ were also used to determine total ADCC killing. These target cells were subsequently used in the ADCC assay as described above (27 (link), 29 (link), 31 (link)). V2 specific ADCC killing was determined by subtracting the ADCC killing in the presence of F(ab’)2 from total ADCC killing of the respective samples.

Rahman M.A., Becerra-Flores M., Patskovsky Y., Silva de Castro I., Bissa M., Basu S., Shen X., Williams L.D., Sarkis S., N’guessan K.F., LaBranche C., Tomaras G.D., Aye P.P., Veazey R., Paquin-Proulx D., Rao M., Franchini G, & Cardozo T. (2023). Cholera toxin B scaffolded, focused SIV V2 epitope elicits antibodies that influence the risk of SIVmac251 acquisition in macaques. Frontiers in Immunology, 14, 1139402.

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Immunoblot Analysis of Protein Samples

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For immunoblot analysis, cell lysates were prepared as described above (Protein biochemistry section). Protein samples were separated using pre-cast 4–12% or 10% Bis Tris Protein Gels (Thermo-Fisher) and standard procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were followed as described in [23 (link)]. A 0.45 μm nitrocellulose membrane (BioRad) was used to transfer proteins from polyacrylamide gels. Commercial antibodies used for immunoblotting were: α-Flag, M2 (Sigma-Aldrich) 1:3,000; α-GFP, JL-8 (Living Colors) 1:5,000; α-Myc, 9E10 (Covance) 1:10,000; A polyclonal α-Dam1c antibody was generated by injecting recombinant Dam1c into rabbits at Pacific Immunology (Ramona, CA). The serum was then used 1:500. The secondary antibodies used were a sheep α-mouse antibody conjugated to horseradish peroxidase (HRP) (GE Life sciences) at a 1:10,000 dilution or a donkey α-rabbit antibody conjugated to HRP (GE Life sciences) at a 1:10,000 dilution. Antibodies were detected using the Super Signal West Dura Chemiluminescent Substrate (Thermo Scientific). For analysis by silver stain, the gels were stained with Silver Quest Staining Kit (Invitrogen).

Gutierrez A., Kim J.O., Umbreit N.T., Asbury C.L., Davis T.N., Miller M.P, & Biggins S. (2020). Cdk1 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments. Current Biology, 30(22), 4491-4499.e5.

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Decorin and Periostin Proteomics

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Samples immunoprecipitated with antibodies against decorin or periostin were subjected to SDS-PAGE, and the gels were stained using the Silver Quest Staining Kit (Invitrogen). The stained gel bands were cut out and treated with dithiothreitol (DTT, Nacalai Tesque, Kyoto, Japan) dissolved in ammonium hydrogen carbonate (Nacalai Tesque), followed by treatment with iodoacetamide (Wako, Osaka, Japan). After the gels were dried, 20 μl of 0.05 pmol/μl trypsin (AB SCIEX) solution was applied to each gel piece and incubated for 12–16 h at 37°C to digest proteins. Digested peptides were extracted by washing the gel pieces twice with 50% trifluoroacetic acid (TFA, Wako), followed by washing with 80% TFA. The purified peptide samples were injected onto a reversed-phase C18 column (HiQ sil C18W-3P, 3 μm, 120 Å; KYA TECH Corp.) and separated by nanoflow liquid chromatography (300 nL/min) on a nano LC Dina-A system (KYA TECH Corp.) in line with a Q-TRAP 5500 instrument (AB SCIEX) using a 75-min gradient of 5–100% acetonitrile in 0.1% formic acid.

Ishiba T., Nagahara M., Nakagawa T., Sato T., Ishikawa T., Uetake H., Sugihara K., Miki Y, & Nakanishi A. (2014). Periostin suppression induces decorin secretion leading to reduced breast cancer cell motility and invasion. Scientific Reports, 4, 7069.

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Protein Gel Electrophoresis and Immunoblotting

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Serum or prot-A^F fractions (volume of 5 μL) were mixed in 1x NuPAGE sample reducing agent (Invitrogen) and 1× NuPAGE LDS sample buffer (Invitrogen) and loaded on NOVEX-NuPAGE 4–12% BT gels (Life technologies, Carlsbad, CA). Protein gels were then silver stained using the SilverQuest staining kit (Invitrogen). For immunoblot, proteins were blotted at 160 mA for 1.5 h onto polyvinylidene difluoride membranes (Millipore) using transfer buffer (Invitrogen). Membranes were blocked with 3% BSA and incubated with HRP-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. Membranes were then rinsed and labeled proteins were detected using the ECL Plus enzymatic chemiluminescence kit (GE Healthcare).

Ratelade J, & Verkman A.S. (2014). Inhibitor(s) of the classical complement pathway in mouse serum limit the utility of mice as experimental models of neuromyelitis optica. Molecular immunology, 62(1), 104-113.

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Analysis of C. burnetii LPS by SDS-PAGE

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LPS samples extracted from C. burnetii phase II antigen were analysed by SDS-PAGE using the NuPAGE^® electrophoresis system (Thermo Fisher Scientific, USA). Briefly, samples were prepared in NuPAGE LDS sample buffer with reducing agent and heated to 70 °C for 10 min prior to loading on a NuPAGE™ 4–12% Bis-Tris gel (Thermo Fisher Scientific, USA). Gels were run in MES SDS running buffer and included an LPS standard and a SeeBlue™ Plus2 Pre-Stained Protein Ladder (Thermo Fisher Scientific, USA). Following electrophoresis, in gel LPS was stained using a SilverQuest™ staining kit (Invitrogen, USA) following the manufacturer’s instructions.

Williams-Macdonald S.E., Mitchell M., Frew D., Palarea-Albaladejo J., Ewing D., Golde W.T., Longbottom D., Nisbet A.J., Livingstone M., Hamilton C.M., Fitzgerald S.F., Buus S., Bach E., Dinkla A., Roest H.J., Koets A.P, & McNeilly T.N. (2023). Efficacy of Phase I and Phase II Coxiella burnetii Bacterin Vaccines in a Pregnant Ewe Challenge Model. Vaccines, 11(3), 511.

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Affinity Purification of Viral Proteins

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AP-MS experiments were performed as previously described12 (link). For protein purification, HEK293 cell lines stably expressing each FLAG-tagged viral protein were divided into two groups. Then, one group was infected with 1 MOI of IAV. After 16 h of infection, 10⁸ cells were collected and lysed in 10 ml of tandem affinity purification buffer4 (link). Cell lysates were precleared with 50 μl of protein A/G resin before the addition of 20 μl of anti-FLAG resin (Sigma, # F2426) and incubation for 16 h at 4 °C on a rotator. The resin was washed three times and transferred to a spin column with 40 μl of 3X FLAG peptide for 1 h at 4 °C on a rotator. The purified complexes were loaded onto a 4–15% NuPAGE gel. The gels were stained with a SilverQuest staining kit (Invitrogen), and lanes were excised for mass spectrometry analysis by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).

Wang L., Fu B., Li W., Patil G., Liu L., Dorf M.E, & Li S. (2017). Comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction. Nature Communications, 8, 13876.

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Immunoprecipitation of Hsp70 from Cancer Cells

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Cancer cells were collected and lysed in a buffer containing 20 mM Tris, pH 7.4, 25 mM NaCl, and 0.1% NP-40. The anti-Hsp70 antibody (BB70; 5 μL) was added to 500 μg of extract together with protein G agarose beads (30 μL) (Upstate), and the mixture was incubated at 4 °C overnight. Samples were washed with lysis buffer and applied to SDS–PAGE. The gel was stained with the SilverQuest staining kit (Invitrogen) (see Figure 4b).

Kang Y., Taldone T., Patel H.J., Patel P.D., Rodina A., Gozman A., Maharaj R., Clement C.C., Patel M.R., Brodsky J.L., Young J.C, & Chiosis G. (2014). Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70. Journal of Medicinal Chemistry, 57(4), 1188-1207.

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