Glycosphingolipids (GSLs) from brain homogenates were extracted with chloroform:methanol (1:2, v/v) overnight at 4°C and further purified using solid-phase C18 columns (Telos, Kinesis, UK). GSLs were dried down under nitrogen and treated with either Cerezyme® (Genzyme, Cambridge, MA) to obtain glucose from GlcCer, or ceramide glycanase (prepared in house from the medicinal leech Hirudo medicinalis/verbena) to obtain oligosaccharides from other GSLs. Liberated glucose and free glycans were then fluorescently-labelled with anthranillic acid (2AA). Excess 2AA label was removed using DPA-6S SPE columns (Supelco, PA, USA). Purified 2AA-labelled glucose and 2AA-labelled glycans were separated and quantified by normal phase high-performance liquid chromatography (NP-HPLC) 34 (link). The NP-HPLC system consisted of a Waters Alliance 2695 separations module and an in-line Waters 2475 multi λ-fluorescence detector set at Ex λ360nm and Em λ425nm. The solid phase used was a 4.6 x 250mm TSK gel-Amide 80 column (Anachem, Luton, UK). Results were normalized to protein content determined using bicinchoninic acid (BCA) assay.
Dpa 6s spe columns
Manufactured by Merck Group
Sourced in United States
The DPA-6S SPE columns from Merck Group are solid-phase extraction (SPE) columns designed for sample preparation and purification. They feature a silica-based sorbent material that enables efficient extraction and concentration of analytes from various sample matrices. The core function of these columns is to facilitate the selective separation, isolation, and enrichment of target compounds prior to analytical testing or further downstream processing.
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3 protocols using dpa 6s spe columns
1
Glycosphingolipid Quantification from Brain
2
Quantitative Brassinosteroid Analysis
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Samples’ BR contents were analyzed as previously described (Oklestkova et al., 2017 (link)) with a few modifications. Briefly, lyophilized samples (40 mg DW) were homogenized to a fine consistency using 3 mm zirconium oxide beads and an MM 301 vibration mill at a frequency of 30 Hz for 3 min (Retsch, Haan, Germany). The samples were then extracted overnight with stirring at 4°C using a benchtop laboratory rotator (Stuart SB3; Bibby Scientific, Cole-Parmer, Staffordshire, United Kingdom) after adding 1 mL of ice-cold 60% acetonitrile and 30 pmol of [2H3]brassinolide (BL), [2H3]castasterone (CS), [2H3]typhasterol (TY), [2H3]24-epiBL, [2H3]24-epiCS, [2H3]28-norBL, and [2H3]28-norCS (OlchemIm, Olomouc, Czech Republic) as internal standards. The samples were then centrifuged, purified using DPA-6S SPE columns (Supelco, Bellefonte, PA, USA) and evaporated to dryness in vacuo. They were then dissolved in 75 μl 100% MeOH with sonication, made up to 1 ml with PBS buffer (pH 7.2) then loaded on an immunoaffinity column (IAC) coated with anti-BR monoclonal antibodies. After washing the IAC with 9 mL H2O, BRs were eluted using 3 mL ice-cold MeOH (-20°C), evaporated in vacuo using a CentriVap® acid-resistant benchtop concentrator (Labconco Corp., MO, USA) and stored at -20°C until analysis.
3
Glycosphingolipid Quantification from Brain
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Glycosphingolipids (GSLs) from brain homogenates were extracted with chloroform:methanol (1:2, v/v) overnight at 4°C and further purified using solid-phase C18 columns (Telos, Kinesis, UK). GSLs were dried down under nitrogen and treated with either Cerezyme® (Genzyme, Cambridge, MA) to obtain glucose from GlcCer, or ceramide glycanase (prepared in house from the medicinal leech Hirudo medicinalis/verbena) to obtain oligosaccharides from other GSLs. Liberated glucose and free glycans were then fluorescently-labelled with anthranillic acid (2AA). Excess 2AA label was removed using DPA-6S SPE columns (Supelco, PA, USA). Purified 2AA-labelled glucose and 2AA-labelled glycans were separated and quantified by normal phase high-performance liquid chromatography (NP-HPLC) 34 (link). The NP-HPLC system consisted of a Waters Alliance 2695 separations module and an in-line Waters 2475 multi λ-fluorescence detector set at Ex λ360nm and Em λ425nm. The solid phase used was a 4.6 x 250mm TSK gel-Amide 80 column (Anachem, Luton, UK). Results were normalized to protein content determined using bicinchoninic acid (BCA) assay.
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