Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl2 containing a protease inhibitor mixture. Protein was determined using the BCA reagent (Thermo Fisher Scientific). Samples were incubated with 15 μM NBD-sphinganine (Avanti Polar Lipids # 810206P), 20 μM defatted BSA (Sigma-Aldrich #10775835001), and 50 μM 16- or 24-fatty acyl-CoA (Avanti Polar Lipids 870743 & 870725) in a 20 μl reaction volume. CerS (40 μg protein, 25 min reaction time) was assayed using C24.1-CoA and Cer5/6 (5 μg protein, 5 min reaction time) assayed using C16-CoA. Reactions were terminated by chloroform/methanol (1:2, v/v) and lipids extracted. Lipids were dried under N2, resuspended in chloroform/methanol (9:1, v/v), and separated by thin layer chromatography using chloroform/methanol, 2M NH4OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon5 imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
Amersham typhoon5 imager
Manufactured by GE Healthcare
Sourced in United Kingdom
The Amersham Typhoon5 imager is a high-performance phosphor imaging system designed for the detection and quantification of radioactive and fluorescent signals in various life science applications. It offers high-resolution image acquisition and analysis capabilities for a wide range of sample types, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Amersham typhoon, by Cytiva (3 mentions)
Imagequant tl, by GE Healthcare (2 mentions)
Bca reagent, by Thermo Fisher Scientific (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680lt, by LI COR (1 mentions)
Fatty acyl coa, by Avanti Polar Lipids (1 mentions)
Defatted bsa, by Merck Group (1 mentions)
Nbd sphinganine, by Avanti Polar Lipids (1 mentions)
6 protocols using amersham typhoon5 imager
1
Quantification of Ceramide Synthase Activity
2
Fluorescence-Based Activity Assay for rhGBA
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rhGBA produced in BEVS[58] was prepared at 500, 250, 100, 50, 10, 1, 0.1 and 0.01 nM in 150 mM McIlvaine buffer pH 5.2 (supplemented with 0.1 % (v/v) Triton X‐100 and 0.2 % (w/v) sodium taurocholate). ABP 6 or ABP 9 were added to 150 nM final concentration and the reactions were incubated at 37 °C for 30 mins. The samples were denatured with Laemmli (x3) sample buffer by heating at 95 °C for 5 minutes and resolved by electrophoresis in 10 % SDS‐PAGE gels, running at 200 V for ∼50 minutes. Wet slab gels were scanned on fluorescence using an Amersham Typhoon 5 Imager (GE Healthcare) with λEX 635 nm; λEM >665 nm.
3
Quantitative Analysis of rhGBA Binding
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rhGBA produced in BEVS[58] was diluted to 200 nM in 150 mM McIlvaine buffer pH 5.2 (containing 0.1 % (v/v) Triton X‐100 and 0.2 % (w/v) sodium taurocholate) and ABP 6 or ABP 9 were added to 150, 100, 50, 10, 1, 0.1, 0.01 or 0.001 nM final concentration. The reactions were incubated at 37 °C for 30 mins and denatured with Laemmli (x3) sample buffer at 95 °C for 5 minutes. The samples were resolved by electrophoresis in 10 % SDS‐PAGE gels, running at 200 V for ∼50 minutes. Wet slab gels were scanned on fluorescence using an Amersham Typhoon 5 Imager (GE Healthcare) with λEX 635 nm; λEM >665 nm.
4
Kinetic Analysis of rhGBA Activity
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rhGBA produced in BEVS[58] was prepared at 700 nM in 150 mM McIlvaine buffer pH 5.2 (containing 0.1 % (v/v) Triton X‐100 and 0.2 % (w/v) sodium taurocholate) and ABP 7 or ABP 8 were added to 150 nM. The reactions were incubated at 37 °C and aliquots were taken at 2, 5, 10, 30 and 60 mins. The aliquots were immediately denatured with Laemmli (x3) sample buffer by heating at 95 °C for 5 minutes. The samples were resolved by electrophoresis in 10 % SDS‐PAGE gels, running at 200 V for ∼50 minutes. Wet slab gels were scanned on fluorescence using an AmershamTyphoon 5 Imager (GE Healthcare) with λEX 635 nm; λEM >665 nm.
5
Quantitative Western Blot Analysis of PlpD
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Proteins were separated by SDS-PAGE on 8–16% Tris-glycine gels (Invitrogen) and transferred to nitrocellulose membranes using an iBlotII (Life Technologies). Blots were probed overnight using a mouse monoclonal anti-StrepII antibody (Qiagen catalog number 34850) at a 1:2500–1:5000 dilution or a rabbit polyclonal antiserum raised against the PlpD C-terminal peptide NH2-EWLTRVQLGDRQELYSEFYQC-COOH at a 1:5000 dilution. Blots were subsequently probed with a fluorescent goat anti-mouse (IRDye 800CW) or goat anti-rabbit (IRDye 680LT) antibodies (Licor Cat. No. 926–32210 or 926–68021, respectively). The membranes were then scanned using an Amersham Typhoon 5 imager (GE Healthcare) with a 685 nm laser (IRshort720BP20 filter) or 785 nm laser (IRlong825BP30 filter) and the PMT set at 450 V or 700 V, respectively. Pixel intensities of detected proteins were measured using Fiji software (v2.9.0/1.53t). Within-lane values were used to calculate the percent intra-molecular disulfide-bond formation [(intraPlpD/ (intraPlpD + interPlpD + FLPlpD)) × 100] or the percent stable fragment resulting from chymotrypsin digestion. Uncropped images of all blots are included in the Source Data file.
6
Sphinganine Acylation Activity Assay
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 containing a protease inhibitor mixture. Protein was determined using the BCA reagent (Thermo Fisher Scientific). Samples were incubated with 15 µM NBD-sphinganine (Avanti Polar Lipids), 20 µM defatted BSA (Sigma-Aldrich), and 50 µM fatty acyl-CoA (Avanti Polar Lipids) in a 20 µl reaction volume. CerS (40 µg protein, 25 min reaction time) was assayed using C24.1-CoA and Cer5/6 (5 µg protein, 5 min reaction time) assayed using C16-CoA. Reactions were terminated by chloroform/methanol (1:2, v/v) and lipids extracted. Lipids were dried under N 2 , resuspended in chloroform/methanol (9:1, v/v), and separated by TLC (Merck) using chloroform/methanol, 2M NH 4 OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon5 imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 containing a protease inhibitor mixture. Protein was determined using the BCA reagent (Thermo Fisher Scientific). Samples were incubated with 15 µM NBD-sphinganine (Avanti Polar Lipids), 20 µM defatted BSA (Sigma-Aldrich), and 50 µM fatty acyl-CoA (Avanti Polar Lipids) in a 20 µl reaction volume. CerS (40 µg protein, 25 min reaction time) was assayed using C24.1-CoA and Cer5/6 (5 µg protein, 5 min reaction time) assayed using C16-CoA. Reactions were terminated by chloroform/methanol (1:2, v/v) and lipids extracted. Lipids were dried under N 2 , resuspended in chloroform/methanol (9:1, v/v), and separated by TLC (Merck) using chloroform/methanol, 2M NH 4 OH (40:10:1, v/v/v) as the developing solvent. NBD-labeled lipids were visualized using an Amersham Typhoon5 imager and quantified by ImageQuantTL (GE Healthcare, Chalfont St Giles, UK). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
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