A total of 10 or 15 μg of total protein from tissue lysates was separated by SDS-PAGE electrophoresis using a Tetra cell (Bio-Rad; Hercules, CA, USA), in 12% polyacrylamide separation gel and a 4% polyacrylamide stacking gel, applying a constant voltage of 120 V. Pre-stained standard proteins and pool sample, composed by 10 µL of cerebellum samples from each mouse, were also loaded onto the gel for inter-gel normalization purposes. Ten micrograms of total protein from tissue lysates were also loaded onto nitrocellulose membranes using a dot-blot system, and the wells washed twice with PBS before removing the membrane from the apparatus.
Tetra cell
Manufactured by Bio-Rad
Sourced in United States
The Tetra cell is a versatile electrophoresis system designed for conducting gel electrophoresis experiments. It is capable of performing a range of electrophoresis techniques, including SDS-PAGE, native PAGE, and agarose gel electrophoresis. The Tetra cell provides a controlled and consistent environment for separating and analyzing biomolecules, such as proteins and nucleic acids.
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Lab products found in correlation
Anti rabbit igg f ab 2 specific, by Jackson ImmunoResearch (2 mentions)
Anti human iga α chain specific, by Merck Group (2 mentions)
Pierce protein free tbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg whole molecule, by Merck Group (2 mentions)
Semi dry transfer apparatus, by Bio-Rad (2 mentions)
Supersignal west dura chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
Semi dry blotting apparatus, by Bio-Rad (1 mentions)
Las 3000 image analyzer, by Fujifilm (1 mentions)
Gel doctm xr, by Bio-Rad (1 mentions)
Mouse anti gfp, by Roche (1 mentions)
Gfp beads, by Proteintech (1 mentions)
Anti ha, by Roche (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Benchmarktm protein ladder, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Histrap excel, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
13 protocols using tetra cell
1
SDS-PAGE Protein Separation and Normalization
2
Immunoblotting of Mouse Serum and Lavage
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Mouse samples (10μl serum diluted 1:1000, or 20μl undiluted nasal lavage) were resolved on a 10% Mini-PROTEAN®TGX™ gel using a Tetra cell (Bio-Rad, Hercules, CA). Reduced samples were prepared with Laemmli sample buffer containing β-mercaptoethanol (βME) and boiled for 5 mins. Non-reduced samples were prepared without βME and boiling. Gels were transferred onto PVDF membranes by semi-dry transfer apparatus (Bio-Rad). Membranes were blocked in Pierce Protein-Free (TBS) Blocking Buffer (Thermo Fisher Scientific). Whole rabbit IgG was detected in lavages using anti-rabbit IgG (whole molecule) (Sigma, St. Louis, MO). Rabbit IgG fragments were detected using anti-rabbit IgG (F(ab’)2 specific) (Jackson ImmunoResearch Laboratories, West Grove, PA). hmAbs were detected using anti-human IgA (α-chain specific) (Sigma).
3
Quantifying Glycosaminoglycan Levels by FACE
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A potential limitation of the HABP ELISA-like assay is the lower sensitivity for detection of very low molecular weight (LMWHA) and oligomeric fragments of HA30 (link). We therefore performed FACE analysis to confirm ELISA results. FACE is a well-validated method to obtain highly accurate picomolar quantitation of glycan species by reductive amination31 (link). Briefly, a 1 µL aliquot of the resuspended glycosaminoglycan pellet (from sample preparation for fragmentation analysis, described below) was digested with excess Streptococcus dysgalactiae hyaluronidase to cleave HA into unit disaccharides. The solution of disaccharides was then lyophilized. The dissacharides were reductively aminated by adding 1 µL/2.5 mg wet tissue of 2-aminoacridone (AMAC, 6.25 mM) in 1:1 mixture with 2 M sodium cyanoborohydride, followed by incubation for 18 hours at 37 °C. The samples were then loaded onto a 40% acrylamide gel and vertically electrophoresed (TetraCell, Bio-Rad) at 5 °C for 1 hour at 500 V. FACE standards, with known concentrations of HA n-mers, were used for calibration. The gels were then transferred to a UV imager for fluorescence quantitation. Absolute HA concentrations were determined by normalization against the HA-2 disaccharide standard, and then against dsDNA loaded as described below.
4
Separated WTA Protein Visualization
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WTA preparations were separated on tricine polyacrylamide gels using a Bio-Rad tetra cell according to a previously described method (Brignoli et al., 2022 (link)). The gels were separated at 4°C using a constant amperage of 40 mA under constant stirring until the dye front reached the bottom. Gels were washed three times in MilliQ H2O followed by staining in 1 mg/ml Alcian blue overnight. Gels were subsequently destained in in MilliQ H2O, until the WTA became visible and finally imaged.
5
Alcian Blue Staining of WTA Separation
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WTA preparations were separated on tricine polyacrylamide gels using a BioRad tetra cell according to a previously described method58 (link). The gels were separated at 4°C using a constant amperage of 40 mA under constant stirring until the dye front reached the bottom. Gels were washed 3 times in MilliQ H2O followed by staining in 1 mg/ml Alcian blue overnight. Gels were subsequently destained in in MilliQ H2O, until the WTA became visible and finally imaged.
6
Western Blot Analysis of Urinary Proteins
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Western blot analysis was conducted on the urine samples. Individual urine samples were concentrated by TCA-precipitation and the concentrations of proteins in the samples were measured by using 2D Quant Kit (GE Healthcare) according to the manufacturer’s instructions. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 12% polyacrylamide gels using a vertical slab gel apparatus under non-reducing conditions (Bio-Rad TetraCell) as described previously [33 (link)]. A total of 5 μg of protein from each sample was loaded into the gel and the proteins were separated by electrophoresis at 100 V for 2 h 30 min. Proteins were transferred to a PVDF membrane (Immobilon, Amersham) using a semi-dry blotting apparatus (Bio-Rad). Nonspecific binding to the membranes was blocked by an incubation for 1 h using 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were thereafter probed with polyclonal anti-C3c in TBST at 4°C. Membranes were then washed with TBST and incubated for 3 h at room temperature with alkaline phosphatase-conjugated anti-rabbit (1:2000, Santa Cruz) in TBS. Proteins were visualized using Super Signal West Dura chemiluminescence substrate (Thermo) and imaged by the LAS3000 image analyzer (Fuji).
7
Recombinant Pichia pastoris Protein Production
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A recombinant P. pastoris GS115 bearing the target gene in its genome was incubated in 100 ml of BMGY until the OD600 reached approximately 20. All cells were harvested by centrifugation and transferred into 50 ml of BMMY. A total of 1 % (v/v) methanol was added every 24 h to induce the expression of the target protein. Approximately 1 ml of cell culture was collected every 24 h and centrifuged at 8000 × g for 5 min to remove cells. After 144 h, an equal volume of each supernatant was loaded onto a 12 % (w/v) polyacrylamide gel for SDS-PAGE, followed by staining with Coomassie brilliant blue G-250. The Western blot assay was implemented using the standard technique. The gels were electroblotted on polyvinylidenefluoride (PVDF) membranes using Tetra Cell (Bio-Rad, USA). For immune detection, we used a primary anti-His murine monoclonal antibody (California Bioscience, USA) and a goat anti-mouse IgG HRP-conjugated secondary antibody (California Bioscience, USA); the blots were visualized using the Bio-Rad Gel DOCTM XR+ (USA) imaging system.
8
Immunoblotting of Mouse Serum and Lavage
9
Separated WTA Protein Visualization
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WTA preparations were separated on tricine polyacrylamide gels using a Bio-Rad tetra cell according to a previously described method (Brignoli et al., 2022 (link)). The gels were separated at 4°C using a constant amperage of 40 mA under constant stirring until the dye front reached the bottom. Gels were washed three times in MilliQ H2O followed by staining in 1 mg/ml Alcian blue overnight. Gels were subsequently destained in in MilliQ H2O, until the WTA became visible and finally imaged.
10
GFP-based Protein Interactome Isolation
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Late third instar larvae (80) and pelleted Kc167 cells were homogenized in lysis buffer (50 mM Tris pH 7.5;150 mM NaCl;1 mM EDTA;1 mM EGTA; 2.5 mM pyrophosphate; 1 mM Na3VO4; 1 mM glycerol phosphate) for 1 h (4°C). Cellular debris was spun at 3,000 rpm for 15 min at 4°C. Supernatant was again spun at 9,500 rpm for 45 min at 4°C. Supernatant was added to equilibrated GFP-beads (Chromo Tek, NY, US) and left rotating overnight at 4°C. Beads were washed several times and then boiled in Laemmi Loading Buffer (Biorad, CA, US). Beads were loaded onto 8% or 12% polyacrylamide gel and proteins were separated by SDS-PAGE (Biorad). Specific proteins were detected by Western Blot using a semidry blotting or tetra cell (Biorad). Antibodies used were: mouse anti-GFP 1:3000 (Roche); anti-HA 1:5000 (Roche); anti-Myc 1:1000 (Upstate).
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