Co-immunoprecipitation experiments using AtTCTP-GFP or AtCSN4-GFP as bait were performed using the μMACS GFP Isolation Kit (Miltenyi Biotec). Three biological replicates were performed for each sample. Wild-type Col-0 and 35S::GFP plants were used as controls. Tissues of 10 days-old seedlings, mature seeds harvested from green siliques or inflorescences were ground to a fine powder in liquid nitrogen using mortar and pestle. The tissue powder (200 mg) was resuspended with 1ml pre-cooled (4°C) Miltenyi lysis buffer complemented with one tablet of cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche) for 5 ml of lysis buffer. Cellular extracts were incubated on ice 10 min and then centrifuged 10 min at 21 000g (4°C). The supernatants were incubated with 50μl anti-GFP antibody coupled to magnetic μMACS microbeads for specific isolation of GFP-tagged protein during 1 hour at 4°C on orbital shaker. Microbeads were bound to magnetic columns and washed as described by the manufacturer, before elution of GFP-tagged proteins and bound proteins. Eluted proteins were analyzed by Western blot. For the immunoprecipitation followed by mass spectrometry (IP/MS) we visualized proteins by silver staining of the SDS-PAGE gel (ProteoSilver Plus Silver Stain Kit, SIGMA).
Proteosilver plus silver stain kit
Manufactured by Merck Group
Sourced in United States
The ProteoSilver Plus Silver Stain Kit is a laboratory product designed for the visualization of proteins in polyacrylamide gels. It is a silver-based staining method that allows for the detection of proteins at nanogram levels. The kit includes all the necessary reagents and solutions for the staining process.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Proteomelab pps system, by Beckman Coulter (2 mentions)
Speedvac concentrator, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Nupage novex 4 12 bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
μmacs gfp isolation kit, by Miltenyi Biotec (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nbt bcip, by Avantor (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Chemiluminescence substrate kit, by Thermo Fisher Scientific (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
Pierce centrifuge column, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
C18 ziptip, by Merck Group (1 mentions)
34 protocols using proteosilver plus silver stain kit
1
Co-immunoprecipitation of GFP-tagged Proteins
2
Protein Quantification and Identification by Mass Spectrometry
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Protein quantification was performed using the Bradford assay according to the supplier's instructions (Bio‐Rad).
Eluates from immunoprecipitation or columns were denatured with Laemmli denaturing buffer and separated by SDS/PAGE using pre‐cast gradient gels with MOPS/SDS running buffer (NuPAGE® Novex®4–12% Bis‐Tris polyacrylamide gel, Invitrogen, Thermo Fisher Scientific). Proteins were either stained on gel using ProteoSilver Plus silver stain Kit (Sigma‐Aldrich), or electrotransferred onto a poly(vinylidene difluoride) membrane (Immobilon‐P; EMD Millipore) and immunodetected by colorimetry (alkaline phosphatase conjugated goat secondary antibodies from Promega, and NBT‐BCIP from Amresco as substrate). For identification by mass spectrometry (MS), proteins were separated by SDS/PAGE (NuPAGE® Novex®4–12% Bis/Tris polyacrylamide gel) in a MES/SDS running buffer (Invitrogen), and stained with Colloidal Blue Staining Kit (Invitrogen).
Eluates from immunoprecipitation or columns were denatured with Laemmli denaturing buffer and separated by SDS/PAGE using pre‐cast gradient gels with MOPS/SDS running buffer (NuPAGE® Novex®4–12% Bis‐Tris polyacrylamide gel, Invitrogen, Thermo Fisher Scientific). Proteins were either stained on gel using ProteoSilver Plus silver stain Kit (Sigma‐Aldrich), or electrotransferred onto a poly(vinylidene difluoride) membrane (Immobilon‐P; EMD Millipore) and immunodetected by colorimetry (alkaline phosphatase conjugated goat secondary antibodies from Promega, and NBT‐BCIP from Amresco as substrate). For identification by mass spectrometry (MS), proteins were separated by SDS/PAGE (NuPAGE® Novex®4–12% Bis/Tris polyacrylamide gel) in a MES/SDS running buffer (Invitrogen), and stained with Colloidal Blue Staining Kit (Invitrogen).
3
Quantitative Analysis of Griffithsin Purity
4
Protein Identification via Mass Spectrometry
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Protein bands were stained using the ProteoSilver Plus Silver Stain Kit (Sigma, St. Louis, MO) according to the manufacturer’s protocol and excised from the polyacrylamide gel. After washing, the gels were digested by incubation in buffer with trypsin [32] (link). The tryptic digests were analyzed by an LC-MS/MS system as described previously [33] (link). Database search was performed using MASCOT software (version 2.2.1., Matrix Science Ltd., London) and the NCBI Refseq sequence database under the parameters as described previously [34] (link).
5
Proteomic Analysis of Extracellular Vesicles
6
SDS-PAGE Analysis of Purified Virus
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The purified virus samples were lysed in loading buffer and resolved by electrophoresis in a 12% SDS-PAGE. After electrophoresis, the gel was fixed with 100 ml of the fixing solution (adding 50 ml of ethanol and 10 ml of acetic acid to 40 ml of ultrapure water) in a clean tray for 20 minutes or longer time. Then the gel was washed by 30% ethanol solution and ultrapure water in sequence. After that, the gel was stained with the ProteoSilver Plus Silver Stain Kit (Sigma) according to the manufacturer’s instructions.
7
Immunoprecipitation and Mass Spectrometry
8
Purification of scFv Antibody Fragments
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The scFv clones were transformed in TOP10 E. coli non-suppressor strain and then used to inoculate SB medium containing 50 μg ml−1 of carbenicillin and 2% glucose. The culture was grown under agitation (250 r.p.m.) overnight at 37 °C and 2 ml were diluted in 200 ml of supplemented SB medium. The culture was incubated under agitation for 6–8 h at 37 °C (OD600=1.0), and centrifuged at 3000 g for 10 min at 4 °C. Bacteria were resuspended in 200 ml supplemented carbenicillin SB medium and induced by 2 mM IPTG at 30 °C overnight. Supernatant containing antibody was obtained after centrifugation at 5000 g , 4 °C, 15 min, after that, His-tagged scFv fragments were purified by immobilized-metal (Ni) affinity chromatography (HisTrap HP, GE Healthcare) by high-performance liquid chromatography (HPLC, Amersham Biosciences, Sunnyvale, CA, USA) according to the manufacturer's instructions. All eluted fractions were identified for the presence of antibody fragments by dot-blot immunoassay, positive fractions were pooled, desalted by a Centriprep column (Millipore, Billerica, MA, USA), lyophilised, resuspended in PBS and then measured at 280 nm for protein. The purity and correct size of scFv was verified on 12% SDS–PAGE gel followed by silver staining (ProteoSilver Plus Silver Stain Kit, Sigma).
9
Haptoglobin Purification and Analysis
10
Isolation and Detection of DNA-Protein Crosslinks
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DPCs were isolated using a modified Rapid Approach to DNA Adduct Recovery (RADAR) assay65 (link),25 (link). Briefly, 1–2 × 106 cells were lysed in 1 mL of buffer containing 6 M guanidine thiocyanate (GTC); 10 mM Tris–HCl, pH 6.8; 20 mM EDTA; 4% Triton-X100; 1% Sarkosyl and 1% DTT. DNA was precipitated by adding 100% ethanol, washed three times in wash buffer (20 mM Tris–HCl, pH 6.8; 150 mM NaCl and 50% ethanol). DNA was then solubilized in 1 mL of 8 mM NaOH. The DNA concentration was quantified by treating a small aliquot of DNA with proteinase K (Invitrogen) for 3 h at 50 °C, followed by detection with PicoGreen dye (Invitrogen) according to manufacturer’s instructions. Equal dsDNA loading was confirmed by slot-blot immunodetection with an anti-dsDNA antibody (Abcam). Total DPCs after electrophoretic separation on polyacrylamide gels were visualised by silver staining using the ProteoSilver Plus Silver Stain Kit (Sigma-Aldrich) and specific proteins were detected by Western-blot.
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