Trypomastigote pellets (3 x 108) were homogenized in 500 μL of ConA buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) Nonidet P40, 0.1% (w/v) sodium deoxycholate, 5 mM Cl2Ca, 5 mM Cl2Mg, 5 mM Cl2Mn, 1% (v/v) inhibitor protease cocktail (Sigma), and 1 mM DTT] and processed as described [36 (link)]. After clarification, parasite extract was fractionated overnight at 4°C onto 100 μL of ConA–Sepharose (GE Healthcare), and retained glycoproteins eluted with 50 μL of SDS-PAGE loading buffer. Flow-through and ConA-bound fractions were analyzed by Western blot with different antisera. For N-glycosylation analysis, trypomastigotes were lysed in 1X Glycoprotein Denaturing Buffer (BioLabs), boiled for 10 min and extracts corresponding to 2.5 x 107 parasites were treated with 2,000–3,000U of Endoglycosidase H for 1 h following manufacturer’s procedures (BioLabs) and analyzed by Western blot.
Con a sepharose
Manufactured by GE Healthcare
Sourced in Sweden
Con A Sepharose is a chromatography resin used for the purification of glycoproteins. It consists of Concanavalin A, a lectin, coupled to Sepharose beads. The Concanavalin A binds to the carbohydrate moieties present on glycoproteins, allowing for their capture and separation from other components in a sample.
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Lab products found in correlation
2 d clean up kit, by GE Healthcare (2 mentions)
Vivaspin concentrator, by Sartorius (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Inertsil ods 3, by GL Sciences (1 mentions)
High performance lc system, by Shimadzu (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Transit lt1, by Mirus Bio (1 mentions)
Enhanced chemi luminescence (ecl), by PerkinElmer (1 mentions)
Econo column chromatography column, by Bio-Rad (1 mentions)
Poly prep column, by Bio-Rad (1 mentions)
10 protocols using con a sepharose
1
Purification and Characterization of Trypomastigote Glycoproteins
2
Purification and Characterization of Alpha-1-Antitrypsin
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PCM (8 ml) was subjected to chromatography on Q Sepharose (GE Healthcare) and eluted with a stepwise pH gradient. The active fractions (pH 4.5 to 3.0) were further subjected to chromatography on Con A Sepharose (GE Healthcare) and eluted with 0.5 M mannoside. The eluted fraction was applied to nano-LC/MS/MS. The immunoprecipitants with anti-A1AT antibody from 24 ml of PCM were treated with 8 M urea and glycine-HCl (pH 2.6). The eluted substances were applied to a C18 column (Inertsil ODS-3, GL Sciences) on a high-performance LC system (Shimadzu). The injection volume was set to 100 μl, and the flow rate was 800 μl/min. The mobile phases consisted of (A) 0.5% acetic acid and (B) 0.5% acetic acid in 80% acetonitrile. Two-step linear gradient programs ranging from 5 to 99% B were used.
3
Glycogen Binding Assay with Con A-Sepharose
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Con A–Sepharose (GE Healthcare) beads were used to assay glycogen binding. Fresh Con A beads were washed in Con A buffer [67 mM Hepes (pH 6.8), 0.2 mM CaCl2, 10 mM MgCl2, 500 mM NaCl, and 4 mM DTT] and incubated with rabbit liver glycogen (50 mg/ml; Sigma-Aldrich, G-8876, 1:1 volume) at 4°C for 1 hour. Glycogen-conjugated Con A beads were washed three times with Con A buffer and resuspended as 50% slurry. Con A–glycogen Sepharose beads (30 μl) were incubated with 15 μg of phosphorylase a, GM64–237, and PP1α7–330 alone and in various combinations thereof in a total volume of 250 μl for 1 hour at 4°C under gentle mixing. The beads were washed three times with Con A buffer (750 μl) and recovered by centrifugation (2000g, 1 min). The supernatant was removed using gel-loading tips, leaving behind the beads that were then incubated with 25 μl of 1 M α-d -methylglucoside (in Con A buffer) to elute all proteins bound to glycogen. Eluate (10 μl) was carefully transferred to new tubes to avoid contamination with residual beads and analyzed by SDS-PAGE. SDS-PAGE was stained using Sypro Ruby (Invitrogen) for visualization of total proteins.
4
Preparation of Recombinant Acid Ceramidase
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A full-length human aCDase or aCDase lacking an inter-subunit disulfide bond (C31A) was inserted into a pcDNA3.1(+) expression vector. These expression vectors or pcDNA3.1(+) itself were transfected into HEK293 cells using Trans-it LT1 (Mirus; Madison, WI, USA). At 48 h post transfection, the medium was collected and mixed with SDS-PAGE sample buffer without reducing agent. aCDase (C31A) was separated into alpha- and beta-subunits by electrophoresis. For biochemical experiments, recombinant aCDase was prepared as follows: The medium containing expressed aCDase was subjected to affinity chromatography using ConA-sepharose (GE healthcare; Little Chalfont, England). The fraction containing aCDase was filtrated using an Ultrafree filtration unit (10,000 MW, Millipore; Billerica, MA, USA) with 10 mM Tris-HCl (pH 7.5) containing 20 mM NaCl.
5
Glycoprotein Enrichment and Purification
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Ten milliliters of harvested medium was mixed with 2 ml of ConA Sepharose (GE Healthcare Bio-Sciences) and rotated overnight at 4°C. The mixture was then loaded into a column (0.8 cm × 4 cm) (Bio-Rad Laboratories, Hercules, CA, USA) and equilibrated with equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4). The column was washed with 50 ml of equilibration buffer to remove non-glycosylated and O-glycosylated proteins, and bound N-glycoproteins were eluted with 0.3 M methyl-α-D-glucopyranoside. Absorbance at 280 nm was measured throughout the chromatographic process. Eluted fractions were concentrated 100 times using Vivaspin concentrators (10,000 molecular weight cut-off; Sartorius) and further desalted with 2-D Clean-Up Kits (GE Healthcare Bio-Sciences) and then subjected to 2-DE analysis.
6
Mouse Organ Nrp1 Expression Analysis
7
Purification of N-Glycoproteins using Con A
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Concanavalin A (Con A) is a tetrameric metalloprotein isolated from Canavalia ensiformis (jack bean). Con A coupled to sepharose is routinely used for the separation and purification of N-glycoproteins. To obtain the chromatographic profiles, 10 ml of harvested medium was mixed with 2 ml of Con-A sepharose (GE Healthcare Bio-Sciences, Uppsala, Sweden) and left overnight at 4oC on a rotator. The mixture was then loaded into a column (0.8 cm X 4 cm; BioRad Laboratories, Hercules, CA, US), equilibrated and washed with equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4) to remove the non-glycosylated and O-glycosylated proteins. The bound N-glycoproteins were then eluted with 0.3 M of methyl-α-D-glucopyranoside. The chromatographic process was monitored at an absorbance of 280 nm. The eluted fractions were concentrated 100 times using Vivaspin 10,000 molecular weight cut-off concentrators. The concentrated fractions were further desalted and subjected to 2D-E analyses.
8
Lectin Chromatography of Glycopeptides
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Aliquots of glycopeptides were subjected to ConA-Sepharose (GE Healthcare, Vélizy Villacoublay, FR) chromatography as described previously [54 (link),55 (link)]. The presence of terminal non-reducing galactose residues on N-glycans was evaluated by subjecting the [2-3H]mannose labeled glycopeptides to Ricinus communis agglutinin I (RCA-I, E.Y Laboratories, San Mateo, CA, USA) lectin chromatography [56 (link)]. Prior to analysis, glycopeptides were treated with 100 µL 50 mM H2SO4 for 1 h at 80 °C. After cooling and neutralization with barium acetate, glycopeptides were applied to the column in 50 mM Tris/HCl (pH 7.8) buffer containing 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 and 0.02% (w/v) sodium azide (LB). The column was subsequently eluted with 10 mL LB (fraction I), 10 mL LB containing 0.1 mM lactose (fraction II), 10 mL LB containing 1.0 mM lactose (fraction III), and finally 10 mL LB containing 10 mM lactose (fraction IV).
9
Affinity Purification of Recombinant APA Protein
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The affinity of the recombinant APA protein for concanavalin was determined by affinity chromatography. ConA Sepharose (GE Healthcare, DE) resin was washed in equilibrium buffer (20 mM Tris–HCl, 0.5 M NaCl, pH 7.4) and 5 mL of resin were incubated with 100 mL of soluble proteins overnight at 4 °C with gentle agitation in a blood homogenizer. The mixture was transferred to an Econo-Column chromatography column of 1.5 × 10 cm (Bio-Rad, USA) and washed with 50 mL of equilibrium buffer, using a flow rate of 1 mL/min to elute non-bound protein. Bound proteins were eluted with 0.01 M borate buffer at pH 7.4 and 0.1 M borate buffer at pH 7.4 under continuous flow (30 mL/h). Eluted protein fractions were collected and stored at − 20 °C until used for silver-staining SDS-PAGE and 2D electrophoresis western blot.
10
N-Glycoprotein Enrichment and Purification
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Ten milliliters of harvested medium was added to 2 mL of Con A Sepharose (GE Healthcare Bio-Sciences, Uppsala, Sweden) and gently shaken overnight at 4°C. The mixture was subsequently loaded into a 0.8 × 4 cm Poly-Prep column (BioRad Laboratories, Hercules, CA, USA) and equilibrated with an equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4). The column was washed with 50 mL of equilibration buffer to remove unbound proteins (nonglycosylated and O-glycosylated proteins); and bound N-glycoproteins were eluted with 0.3 M methyl-α-D-glucopyranoside. Chromatographic process was monitored at the absorbance of 280 nm. The eluted fractions were pooled and concentrated 100-fold using Vivaspin concentrators (10,000 molecular weight cut-off; Sartorius). Concentrated eluate was further desalted using 2D Clean-Up Kit (GE Healthcare Bio-Sciences, Uppsala, Sweden) and subjected to 2D-E.
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