The Indomethacin used in this study was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) which was suspended in carboxy-methyl cellulose (CMC) for oral gavage. Criterion precast polyacrylamide gels, TGS and XT MES electrophoresis running buffers, Ready Strip™ IPG strips, mineral oil, dithiothreitol (DTT), iodoacetamide (IA), CHAPS, Biolytes and urea were purchased from Bio-RAD (Hercules, CA, USA).
Biolytes
Manufactured by Bio-Rad
Sourced in United States
Biolytes is a product line of electrolyte measurement solutions offered by Bio-Rad. The core function of Biolytes is to provide accurate and reliable analysis of electrolyte levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Bio-Rad (2 mentions)
Chaps, by Bio-Rad (2 mentions)
Readystrip ipg strip, by Bio-Rad (1 mentions)
Criterion precast polyacrylamide gel, by Bio-Rad (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Urea, by Bio-Rad (1 mentions)
Iodoacetamide, by Bio-Rad (1 mentions)
Mineral oil, by Bio-Rad (1 mentions)
Protean ief cell, by Bio-Rad (1 mentions)
Readystrip, by Bio-Rad (1 mentions)
Ipg strip, by Bio-Rad (1 mentions)
Silver stain plus kit, by Bio-Rad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Clean up kit, by GE Healthcare (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Destreak reagent, by GE Healthcare (1 mentions)
21 g needle, by Terumo (1 mentions)
6 protocols using biolytes
1
Indomethacin Suspension Preparation
2
Isoelectric Focusing and SDS-PAGE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the three replicates of each condition, either for the total proteins or for the membrane proteins, 600 µg of proteins were solubilized into 300 µL of the solubilization buffer supplemented by 1X Biolytes, pH 3-10 (Bio-Rad Laboratories, France) and 0.005% (w: v) bromophenol blue. The protein solution was loaded onto a 17 cm ReadyStrip, pH range 4–7 or 5–8 (Bio-Rad Laboratories, France) according to the manufacturer’s instructions. After an overnight active rehydration step at 50 V in the Protean IEF Cell (Bio-Rad Laboratories) thermo-regulated at 20°C, the isoelectrofocalisation, the SDS-PAGE, and the gel staining were performed as described in the MIAPE GE (Table S1 ).
3
2D Gel Electrophoresis of RPE Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The conditions for strip rehydration, focusing, equilibration, and 2D gel electrophoresis were performed as outlined [11 (link)]. RPE protein (125 μg) were dissolved in a rehydration solution (9 M urea, 3 M thiourea, 6% CHAPS, 1% ASB-14, 1% Biolytes pH 3–10 (Bio-Rad), and 50 mM dithiothreitol) and incubated with 11 cm IPG strips (pH 5 to 8 linear gradient) (Bio-Rad, Hercules, CA, USA). Proteins separated in the first dimension were resolved on 12% polyacrylamide gels. The 2D gels were stained with Flamingo™ Fluorescent Gel Stain according to the manufacturer's protocol. Gels for mass spectrometry were stained with silver using a mass spectrometry-compatible kit (Silver Stain Plus Kit; Bio-Rad).
4
Chloroplast Protein Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated chloroplasts were ground in liquid nitrogen, and the powders were suspended in 20 mL homogenization buffer (see above section) at 4 °C for 1 h. Then trichloroacetic acid was added to the final concentration as 15% (w/v), and stayed at 4 °C for 1 h. After centrifugation (40000 g) at 4 °C for 1 h, the supernatant was removed, and the pellet was washed with acetone containing 1 mM PMSF and 0.07% w/v β-mercaptoethanol twice. The pellet was vacuum dried at −40 °C using a FREEZONE 6 freeze dry system (LABCONCO, USA), and dissolved in 400 μl protein lysis solution containing 7 M urea, 2 M thiourea, 4% w/v CHAPS, 65 mM DTT, 1 mM PMSF and 0.5% v/v biolytes (Bio-Rad). Insoluble materials were removed by centrifugation, and the protein concentration of the sample quantified using the Bradford method28 (link).
5
Protein Extraction from Bacterial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from 15-mL aliquots. Cells were harvested in mid-exponential phase and collected by centrifugation (10,000 × g, 5 min). After washing 3 times with ultrapure water, the cells were lysed in 1 mL buffer containing 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), and 1% protease inhibitor, and the solution was sonicated on ice for 40 min, followed by centrifugation at 20,000 × g for 30 min. The proteins in the supernatant were treated with the Clean-up Kit (GE Healthcare) and dissolved in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% (w/v) dithiothreitol (DTT), 0.5% (v/v) biolytes pH range 3–10 (Bio-Rad), 0.001% (w/v) bromophenol blue). Protein concentration was determined using the BioRad Protein Assay Kit (Bio-Rad) with BSA standard.
6
Biotinylation and Extraction of Primary CD4+ T Cell Surface Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary human CD4 + T cells (5 x 10 6 cells) were washed three times in ice cold PBS (1mL; pH 8) and cell surface proteins labelled with 0.5 mg/mL biotin (Sulfo-NHS-SS-Biotin, Thermo Scientific, UK) for 20 minutes at 4 ºC on a rotary mixer. Cells were washed twice with glycine (10 mM) to quench the biotinylation reaction and washed once with PBS (pH 8). Cells were lysed on ice for 30 minutes in MNE (25mM MES (pH 6.5), 150mM NaCl, 2mM EDTA) lysis buffer supplemented with 1mM sodium orthovanadate, 1% v/v Triton X-100 and 0.1% v/v protease inhibitor cocktail (Sigma Aldrich, UK), genomic DNA was sheared using a 21 G needle (Terumo, UK) and centrifuged at 2,000 xg for 5 minutes to obtain a post-nuclear supernatant (PNS). The PNS (1mL) was incubated for 30 minutes at room temperature with 200 µL of pre washed Magnabind Streptavidin (SA) beads (Thermo Scientific, UK), an unbound fraction was collected and beads were washed twice in MNE lysis buffer and twice in PBS (pH 8). Cell surface proteins were eluted into extraction buffer (8M urea, 2M thiourea, 2% w/v CHAPS, 1% v/v DeStreak reagent (GE Healthcare, Amersham, UK) and 0.2% v/v Bio-Lytes (Bio-Rad)) for 30 minutes at room temperature. PNS, unbound and elution fractions were stored at -80 ºC for SDS-PAGE analysis and western blotting. The yield of CD4+ T cell membrane proteins eluted ranged from 8-20µg for the 19 subjects studied.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!