Overexpression and purification of the proteins of interest from E. coli BL21-Gold (DE3) for structural and biochemical studies were done as described in our study of WhiB7 (26 (link)). Samples after each step of purification were analyzed by SDS-PAGE and by UV-Visible (UV-Vis) spectroscopy. Unless otherwise specified, the final purified proteins in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 0.2 mM tris(2-carboxyethyl)phosphine were stored in liquid nitrogen until use. UV-Vis spectra of the purified proteins were recorded using an HP 8452a diode array UV-Vis spectrophotometer (Agilent Technologies Inc). The absorption at 410 nm, characteristic of proteins containing [4Fe-4S]2+ clusters (43 (link), 44 (link)), was used to estimate the occupancy of the Fe–S cluster in the protein samples containing a Wbl protein. Protein concentrations were estimated either by the Pierce Bradford Assay Kit (Thermo Fisher Scientific) or UV-Vis absorption spectroscopy.
Pierce bradford assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Australia
The Pierce Bradford Assay Kit is a colorimetric assay used for the determination of protein concentration. It utilizes the Bradford method, which is based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. The binding of the dye to the protein results in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Triglyceride quantification kit, by Merck Group (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Pierce trypsin protease, by Thermo Fisher Scientific (1 mentions)
Oasis hlb cartridge, by Waters Corporation (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce high ph reversed phase peptide fractionation kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using pierce bradford assay kit
1
Overexpression and Purification of Wbl Proteins
2
Triglyceride Quantification in Whole Body
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TAG quantification was performed using Triglyceride Quantification kit (MAK266, Sigma-Aldrich). For TAG quantification of whole body, 8 males were used in triplicates for each sample. Triglyceride standard solution and glycerol standard solution were used as standards. Briefly, whole males were homogenized in 100μl solution of 5% NP40 solution and heated for 2 min at 80–100°C twice followed by centrifugation for 2 minutes. The resulting solution was diluted 10 times with water before the assay. 10μl of the solution was used in duplicates following dilution with triglyceride assay buffer (up to 50μl). The samples along with Triglyceride standard were incubated with 2μl lipase in 96-well plates for 20 mins at room temperature. Finally, 50μl of master reaction mixture consisting of triglyceride probe, triglyceride enzyme mix and triglyceride assay buffer was added to the samples and incubated at room temperature for 30–60 minutes and the absorbance was measured at 570nm. TAG levels were determined according to the standard curve. Total protein level in the samples were determined by Pierce Bradford Assay kit (Thermo Scientific).
3
Whole-body Glucose Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glucose quantification was performed using glucose assay kit (MAK263, Sigma-Aldrich) from whole bodies of triplicates of 8 males. The males were homogenized in 100μl of PBS. 6μl of the homogenate in duplicates diluted with Assay buffer (up to 50μl) was used for each sample. 50μl of master reaction mix consisting of glucose assay buffer, glucose probe, and glucose enzyme mix was added to the sample and incubated for 30 minutes at 37°C in dark. Absorbance was measured at 570nm. Glucose levels were determined according to the standard curve. Total protein level in the samples were determined by Pierce Bradford Assay kit (Thermo Scientific) and used for normalization.
4
Purification of Selenomethionine-Substituted Proteins
5
Proteomic Analysis of EE2 Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from control and labelled NT2/D1 cells treated with EE2 for either 30 min (n = 4) or 48 h (n = 3). Each sample was lysed in RIPA buffer (25 mM Tris-HCl pH7.4, 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton X-100) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Sydney, Australia). Lysates were cleared by centrifugation at 13,000 rpm for 20 min at 4 °C. Proteins were precipitated with acetone, and subsequently re-dissolved in digestion buffer (8 M urea in 50 mM TEAB [pH 8.0]). Protein concentrations were measured using a Pierce Bradford Assay Kit (Thermo Fisher Scientific, Sydney, Australia). Protein samples (1 mg) from corresponding control and EE2 treated cells were mixed 1:1 and reduced, alkylated and digested (Pierce Trypsin Protease, Thermo Scientific, Sydney, Australia). An aliquot of 100 μg of the digested peptides were fractionated into 8 fractions using Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher Scientific, Sydney, Australia) and used to determine the global proteome. The remaining peptides were acidified and purified by solid phased extraction using Oasis HLB cartridges (Waters, Sydney, Australia) and used for phosphopeptide enrichment using a titanium dioxide (TiO2) enrichment method [62 (link)]. Eluted phosphorylated peptides were freeze-dried before LC-MS/MS analysis.
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!