EVs were purified from partially engorged I. scapularis salivary glands and uninfected or A. phagocytophilum (MOI 10 and 50)-infected tick ISE6 and IDE12 cells. Protein concentrations in EVs were determined with the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific). Proteins were loaded at an equal amount and electrophoresed in 4–15% Mini-PROTEAN® TGX™ precast gels (Bio-Rad) for 1.5 h at 100 V followed by glycosylation assay with Pierce Glycoprotein Staining Kit (Thermo Fisher Scientific).
Pierce glycoprotein staining kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Glycoprotein Staining Kit is a laboratory product designed to detect and visualize glycoproteins in gel electrophoresis applications. It provides a simple, sensitive, and reliable method for staining glycoproteins in polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pngase f, by New England Biolabs (3 mentions)
Instantblue protein stain, by Abcam (3 mentions)
Endo h, by New England Biolabs (2 mentions)
Bovine liver glycogen, by Merck Group (2 mentions)
Mes sds running buffer, by Thermo Fisher Scientific (2 mentions)
Human salivary α amylase, by Merck Group (2 mentions)
Flamingo fluorescent protein stain, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ab108308, by Abcam (2 mentions)
Dynabeads protein g magnetic beads, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Coomassie blue r 250, by Merck Group (1 mentions)
Histrap ff crude, by GE Healthcare (1 mentions)
Freezedryer epsilon 1 6d, by Martin Christ (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Complete mini protease inhibitor mixture tablet, by Roche (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Mini protean 2 system, by Bio-Rad (1 mentions)
45 protocols using pierce glycoprotein staining kit
1
Purification and Glycosylation Analysis of Tick-Derived EVs
2
Recombinant Enzyme Purification from Methanol-Induced Culture
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After 7 days of methanol-induced culture, the cell-free culture supernatant was collected by centrifugation at 8000 rpm for 15 min. Then, the supernatant was precipitated with ammonium sulfate at 80% saturation and 4 °C overnight. The suspension was centrifuged at 8000 rpm for 15 min and the precipitate was dissolved in 20 mM phosphate buffer solution (pH 7.4). Afterwards, His-tagged recombinant enzymes were purified using Ni2+ affinity chromatography (HisTrap™ FF crude; GE Healthcare, Buckinghamshire, UK), as previously described [49 (link)]. Protein concentrations were estimated using a Pierce™ BCA Protein Assay Kit (Thermo Scientific). SDS-PAGE analysis was carried out in a 12% (w/v) polyacrylamide gel, and staining was conducted with Coomassie blue R-250 (Sigma-Aldrich, St. Louis, MO, USA) and a Pierce™ Glycoprotein Staining Kit (Thermo Scientific), respectively. PNGase F, which is the most effective enzyme for specifically removing N-linked glycans (but not O-linked glycans) from glycoproteins [51 (link)], was obtained from New England Biolabs (Ipswich, MA, USA).
3
Glycoprotein Detection Assay via SDS-PAGE
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For glycoprotein detection assay, SDS-PAGE fractionated purified recombinant proteins were stained using the Pierce Glycoprotein Staining Kit (Thermo Scientific, USA).
4
Glycation of Bovine Serum Albumin
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Glycation of BSA was conducted following a modified protocol from Ledesma-Osuna et al.38 (link). In brief, 5 ml of BSA (20 mg/ml) was mixed with a 5 ml saccharose solution (40 mg/ml), followed by addition of 5 ml 0.1 M phosphate buffer, pH 8.0. Samples were frozen in liquid nitrogen and freeze-dried (FreezeDryer Epsilon 1–6D, Christ, Osterode am Harz, Germany) over night. Samples were then heated to 60 °C for 30, 60 and 120 min respectively. Glycosylation was determined using a periodic acid-schiff reagent method, following the manufacturer’s instructions (Pierce™ Glycoprotein Staining Kit, ThermoFisher Scientific, Waltham, USA), resulting in magenta colored bands of the glycosylated protein.
5
Glycoprotein Staining and Visualization
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Glycoprotein staining was performed using the Thermo Scientific Pierce Glycoprotein Staining Kit in accordance with the manufacturer’s recommendations. An equal amount of each sample was loaded onto two 12% (w/v) polyacrylamide gels. After SDS-PAGE, one gel was stained with Coomassie Brilliant Blue G-250, and the other was stained using the Glycoprotein Staining Kit.
6
Detect Glycoproteins by PAS Method
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After separation on 10% SDS-PAGE, the sugar molecules in ChuaMOX were detected by the periodic acid-Schiff (PAS) method using a Pierce Glycoprotein Staining Kit (Thermo Fisher Scientific).
7
Purification and Glycoprotein Analysis
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Apx toxin fragments were purified by Ni-NTA affinity chromatography, protein concentration was quantified using Bradford reagent (Sigma) and equal amounts of protein were resolved by SDS-PAGE. Gels were fixed in 50% (v/v) methanol and PAS staining was performed using the Pierce Glycoprotein Staining Kit (ThermoFisher) according to the manufacturer’s instructions.
8
Glycoprotein Characterization Using Mass Spectrometry
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Bl-Eng2 protein glycosylation was assessed using the Pierce Glycoprotein Staining Kit (Thermo Fisher Scientific). Monosaccharide composition was determined by gas chromatography as described elsewhere [38 (link)].
9
Glycoprotein Analysis in C. albicans
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C. albicans cells were grown in SC-Mn and SC + Mn for the RNA-seq experiments. Cells were harvested by centrifugation and lysed by bead beating in IP150 buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, and 5% glycerol) supplemented with Complete Mini protease inhibitor mixture tablet (Roche Applied Science) and 10 mM phenylmethylsulfonyl fluoride. The lysates were then cleared by centrifugation, and protein concentration was estimated using the Bradford assay. A total of 80 µg of proteins were heated for 5 min at 95°C in the 2X Laemmli buffer and loaded in an Acrylamide gel. Gels have been stained with silver to observe the total protein profile or with Pierce Glycoprotein Staining Kit (Thermo Fisher) to detect glycoprotein sugar moieties according to the manufacturer’s instructions.
10
Evaluating Collagen Purity and Molecular Weight
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To evaluate protein molecular weight and purity, SDS-PAGE was performed using reagents from the SDS-PAGE Gel Preparation Kit and cast on a Bio-Rad Mini Protean II System (Bio-Rad, Hercules, CA, USA). Freeze-dried collagen solubilized in deionized water (dH2O) at 1 mg/mL was mixed with loading buffer (1:1 v/v) and heated for 2 min at 100 °C to denature the proteins. The SDS gel was composed of 7.5% separation and 4% stacking gel and was loaded with 20 μg of each collagen sample as well as 4 μL of protein marker (Page Ruler Prestained protein ladder, 10 to 250 kDa—Thermo Fisher Scientific, Waltham, MA, USA). After electrophoresis at 90 V, Glycoprotein Stain (Pierce® Glycoprotein Staining Kit—Thermo Fisher Scientific, Waltham, MA, USA) was performed according to the manufacturer’s instructions to stain glycosylated proteins.
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