Protein concentration was determined from the absorbance at 280 nm using the NanoDrop 2000 Spectrophotometer (Thermo Fisher) and the molar extinction coefficient calculated by ExPASy (provided by the Swiss Institute of Bioinformatics). SDS–PAGE protein gels were run on 18-well 10% Criterion XT Bis-Tris Protein Gels (Bio-Rad) at 180 V for 40–60 min. Running buffer (2×) for protein gels was made in-house and contained: 4% sodium dodecyl sulfate (Millipore Sigma), 0.004% bromphenol blue (Sigma-Aldrich), 20% glycerol (Chem-Impex) and 120 mM of Tris HCl (Sigma-Aldrich) at pH 6.8. For reduced gels, SDS–PAGE buffer additionally contained 10% 2-mercaptoethanol and gels were heated to 95 °C for 5 min before gel loading. Protein gels were stained with AcquaStain Protein Gel Stain (Bulldog Bio) and imaged on a LI-COR Odyssey CLx imaging system. Protein sequences are reported in Supplementary Note 1 .
Acquastain protein gel stain
Manufactured by Bulldog Bio
AcquaStain Protein Gel Stain is a ready-to-use solution for staining proteins in polyacrylamide gels. It is a non-toxic, water-based dye that binds to proteins and produces a visible stain, allowing for the detection and analysis of protein bands in gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Tris acetate protein gels, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase conjugated goat anti biotin, by Vector Laboratories (2 mentions)
Biotinylated sambucus nigra lectin sna, by Vector Laboratories (2 mentions)
Immobilon psq polyvinylidene difluoride membranes, by Merck Group (2 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Bromphenol blue, by Merck Group (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Criterion xt bis tris protein gel, by Bio-Rad (1 mentions)
Odyssey clx near infrared fluorescence imaging system, by LI COR (1 mentions)
Xt mops, by Bio-Rad (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Complete his tag purification resin, by Roche (1 mentions)
Talon disposable gravity column, by Takara Bio (1 mentions)
Bead beater, by Biospec (1 mentions)
9 protocols using acquastain protein gel stain
1
Protein Quantification and SDS-PAGE Analysis
2
Lectin Blotting to Detect Terminal α2,6 Sialic Acids
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Equal amounts of SiaIgE or AsIgE were resolved on 3–8% Tris-Acetate protein gels (Life Technologies) in SDS-PAGE under nonreducing conditions. For protein stain, gels were incubated in AcquaStain Protein Gel Stain (Bulldog Bio) for 1 h at room temperature and destained in distilled water. For lectin blotting, the protocol was conducted as described17 (link). Briefly, after resolved proteins on the gel were transferred to Immobilon-PSQ polyvinylidene difluoride membranes (Millipore Sigma), the membranes were blocked with 0.2% BSA in TBS for 1 hour at room temp, washed in TBS, followed by incubation with biotinylated Sambucus nigra lectin (SNA; 0.4 μg/ml; Vector Laboratories) in TBS with 0.1 M Ca2+ and 0.1 M Mg2+ for 1 hour at room temp to determine the level of terminal α2,6 sialic acids on N-linked glycans of proteins. The membrane was then washed in TBS and incubated with alkaline phosphatase conjugated goat anti-biotin (1:5000 dilution; Vector Laboratories) in TBS for 1 hour at room temp. Sialylated proteins on membranes were visualized by incubation with 1-Step NBT/BCIP plus Suppressor Substrate Solution.
3
Purification of TRP47 GST-Fusion Protein
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Plasmids encoding full-length and tandem repeat region TRP47 GST-fusion constructs were transformed into TurboCells BL21(DE3) E. coli and plated on LB agar with ampicillin. Colonies were PCR-screened to verify presence of the insert, and positive transformants were regrown overnight in LB broth with ampicillin. Overnight cultures were diluted 1:20 in fresh growth medium and incubated with shaking at 37°C until an OD600 of about 0.5 was achieved. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich) for 4 h, after which cultures were pelleted and stored at -80°C. Cell pellets were resuspended in Tris-HCl buffer and lysed by sonication in the presence of protease inhibitors (cOmplete mini, EDTA-free; Roche, Basel, Switzerland). Lysates were cleared by centrifugation at 12,000xg and 4°C for 30 min, and the supernatant was collected and incubated with Glutathione Sepharose 4B affinity resin (GE Healthcare Life Sciences) at 4°C with nutation overnight. After washing with Tris-HCl, bound protein was eluted from the resin with 50 mM glutathione. Dialysis was performed and the concentration measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Recombinant protein expression and purification were verified using SDS-PAGE and protein staining (AcquaStain Protein Gel Stain; Bulldog Bio, Portsmouth, N.H.).
4
Mutant Integrase Protein Purification
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Y99H/A128T mutations were introduced in the full-length IN, CCD and CTD-CCD constructs by PCR-directed mutagenesis and the proteins were expressed in BL21 (DE3). Full-length IN and CCD proteins were purified as described (11 (link)). The CTD-CCD proteins were prepared as described (32 (link)). Purified proteins were examined using NuPAGE Bis-Tris 4–12% acrylamide gels with MES as the running buffer (Invitrogen). Proteins were stained AcquaStain Protein Gel Stain (Bulldog-Bio).
5
SDS-PAGE Analysis of Protein Samples
6
Lectin Blotting to Detect Terminal α2,6 Sialic Acids
7
Protein Purity Analysis by SDS-PAGE
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Lyophilized protein was resuspended in phosphate buffer saline (PBS) for further analysis. SDS-PAGE was performed to check the size and purity of the protein. Each sample was heated with NuPAGE LDS sample buffer (Invitrogen, Waltham, MA, USA) at 95 °C before loading into precast Bis-tris 4–12% polyacrylamide gradient gel (Thermo Fisher, Waltham, MA, USA). The gel was stained with AcquaStain Protein Gel Stain (Bulldog Bio. Inc, Portsmouth, NH, USA) according to the manufacturer’s protocol and visualized under white light on a BioRad Chemidoc XRS imaging system.
8
Glycoprotein Sialylation Assay
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Digests against IRDye 800CW-MUC2 (2.25 μg or 4.5 μg) were performed with varying concentrations of AM0627 ± 1 U of SialEXO (Genovis) in PBS (12 μl of total volume) for 22 h at 37 °C. Reactions were loaded onto a 4 to 12% Criterion XT Bis-Tris protein gel (Bio-Rad) and run in XT-MOPS (Bio-Rad) at 180 V. Gels were imaged using an Odyssey CLx Near-Infrared Fluorescence Imaging System (LI-COR Biosciences).
Recombinantly expressed podocalyxin, MUC16, PSGL-1, and CD43 were purchased from R&D Systems (1658-PD, 5609-MU, 3345-PS, and 9680-CD, respectively). C1 esterase inhibitor purified from human plasma was purchased from Innovative Research (IHUC1INH1MG). Fetuin was purchased from Promega (V4961). Digests against recombinant and purified substrates (1 μg) were performed with 50 nM AM0627 or AM0627 mutants ± 1 U of SialEXO in PBS (9 μl of total volume) for 22 h at 37 °C. Reactions were loaded onto a 4 to 12% Criterion XT Bis-Tris protein gel and run in XT-MOPS at 180 V. Protein was visualized with Acquastain Protein Gel Stain (Bulldog-Bio).
Recombinantly expressed podocalyxin, MUC16, PSGL-1, and CD43 were purchased from R&D Systems (1658-PD, 5609-MU, 3345-PS, and 9680-CD, respectively). C1 esterase inhibitor purified from human plasma was purchased from Innovative Research (IHUC1INH1MG). Fetuin was purchased from Promega (V4961). Digests against recombinant and purified substrates (1 μg) were performed with 50 nM AM0627 or AM0627 mutants ± 1 U of SialEXO in PBS (9 μl of total volume) for 22 h at 37 °C. Reactions were loaded onto a 4 to 12% Criterion XT Bis-Tris protein gel and run in XT-MOPS at 180 V. Protein was visualized with Acquastain Protein Gel Stain (Bulldog-Bio).
9
Recombinant His-tagged Protein Purification
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Escherichia coli TOP10 transformed with either pBAD/Thio-TOPO expression vector containing the full-length TRP120 gene upstream of a 6×His tag (55 (link)) or empty vector was inoculated into Terrific Broth (TB) (Corning Inc., Corning, NY), grown overnight, and diluted 1:50 in fresh TB. Cultures were induced with 20% arabinose for 3 h and harvested by centrifuging at 4,000 × g for 20 min at 4°C. The pellets were suspended in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM dithiothreitol [DTT], 2 mM MgCl2, 5 mM EDTA, 5 mM imidazole) and homogenized using a BeadBeater (BioSpec, Bartlesville, OK). Lysate was collected by ice-cold centrifugation at 12,000 × g, run over 1 ml of cOmplete His-tag purification resin (Roche, Basel, Switzerland) packed in a Talon disposable gravity column prepared per manufacturer’s protocol (TaKaRa Bio, Mountain View, CA), and washed with a series of increasingly concentrated imidazole solution washes (5 mM to 40 mM imidazole; 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT, 2 mM MgCl2, 5 mM EDTA). Recombinant protein was sequentially eluted with a 500 mM imidazole solution. Eluate samples were analyzed for purity by SDS-PAGE followed by AcquaStain protein gel stain (BulldogBio, Portsmouth, NH), and then eluates were dialyzed for 24 h in ice-cold PBS and quantified by bicinchoninic acid assay (Thermo Fisher).
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