Pre-cast gradient gels and PVDF membrane-filter paper sandwiches were from Novex; X-OMAT AR film was from Kodak; Rainbow Marker was from Amersham; mannitol, sucrose, EGTA, MgCl2, ATP, ADP, defatted BSA, cytochrome c, HClhCZIBPBHhf-iFeNQq/" target="_blank">oligomycin, Triton and Protease Inhibitor Cocktail were purchased from Sigma. KCl, KOH, HCl, MgSO4, HEPES and Tris were from Fisher Scientific. Calcium Green 5N was purchased from Molecular Probes.
Defatted bsa
Manufactured by Merck Group
Sourced in United States
Defatted Bovine Serum Albumin (Defatted BSA) is a laboratory-grade protein product derived from bovine serum. It has been processed to remove lipids and other impurities, resulting in a highly purified protein preparation. Defatted BSA is commonly used as a stabilizing agent, blocking buffer, and protein standard in various analytical and experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fatty acyl coa, by Avanti Polar Lipids (2 mentions)
X omat ar film, by Kodak (1 mentions)
Rainbow marker, by Cytiva (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (1 mentions)
Mgso4, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Triton, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Polyethyleneimine, by Merck Group (1 mentions)
Silica gel 60 tlc plate, by Merck Group (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Ecl detection system, by Cyanagen (1 mentions)
5 protocols using defatted bsa
1
Mitochondrial Membrane Protein Analysis
2
Lipid Extraction and Analysis Protocol
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NBD-Sphinganine (NBD-Sph) and fatty acyl-CoAs were from Avanti Polar Lipids (Alabaster, AL). Defatted BSA, a protease inhibitor mixture, and polyethyleneimine were from Sigma. An ECL detection system and a BCA reagent kit were from Cyanagen (Bologna, Italy). Silica gel 60 TLC plates were from Merck (Billerica, MA). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).
3
GRP78 Expression in Tunicamycin-Treated L1210 Cells
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L1210 cells were cultivated in the absence or presence of tunicamycin (0.1 μM) for 24 h. Subsequently, the cells were washed three times with PBS, resuspended (5 × 105 cells per mL) in RPMI medium with 5% of defatted BSA (Sigma Aldrich, St. Louis, MO, USA), and incubated for 30 min with anti-GRP78 antibody (described in previous chapter) in a humidified atmosphere supplemented with 5% CO2 at 37 °C. After incubation, the cells were washed three times with RPMI medium containing 5% BSA and subsequently left to interact with secondary antibody (Goat anti-Rabbit IgG linked with Alexa Fluor 660, A21074, Thermo Fisher Scientific, Bremen, Germany). The labeled cells were analyzed on BD Accuri C6 flow cytometer.
4
Antibody Characterization for T. brucei Research
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All custom antibodies have been described previously. The rabbit anti-TbMORN1 were made for a previous project [24 (link)]. The mouse monoclonal anti-Ty1 (BB2) antibodies were a gift from Cynthia He (University of Singapore) [30 (link)]. The mouse monoclonal anti-PFR1,2 antibodies (L13D6) were a gift from Keith Gull (University of Oxford) [31 (link)]. The anti-BiP antibodies were a gift from Jay Bangs (University at Buffalo) [32 (link)]. The rabbit anti-GFP antibodies were a gift from Graham Warren (MRC Laboratory for Molecular Cell Biology) [33 (link)]. The following antibodies were obtained from commercial sources: anti-strep tag StrepMAB-Classic (IBA Biosciences), HRP-conjugated anti-mouse (Thermo Fisher Scientific), anti-GST (Santa Cruz Biotechnology). Defatted BSA was purchased from Sigma-Aldrich.
5
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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