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Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
The Bradford dye-binding assay is a colorimetric assay for measuring total protein concentration. It involves the binding of Coomassie brilliant blue to protein. There is no interference from cations or anions from carbohydrates such as sucrose. However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, as well as strongly alkaline solutions, can interfere with the assay.
Quality and lifecycle management
Published in Sarfaraz K. Niazi, Biosimilars and Interchangeable Biologics, 2016
The methods of choice are as follows: The bicinchinonic acid assay (BCA) is a copper based spectrophotometric assay often preferred to the biuret or Lowry assay due to its simplicity and ruggedness toward many buffer components. The accuracy is good, but protein-to-protein differences in reactivity can occur. Major interfering agents are strong acids, ammonium sulfate, and lipids.The copper based biuret assay is among the oldest of the total protein assays offering low sensitivity (1–10 mg/mL) and still used frequently in more concentrated solutions. The major interfering agents are ammonium salts.The Bradford assay uses the ability of the Coomassie brilliant blue G-250 dye to bind to peptides and proteins. Upon binding the dye undergoes a color shift from 465 to 595 nm. This assay can be used in dilute solutions.The Lowry assay and its modifications (Hartree–Lowry) are enhancements of the biuret reaction making use of the Folin-Ciocalteau reagent. The color reaction is time dependent and protein-to-protein variations may occur. The accuracy is good. Numerous buffer components can interfere with the assay (e.g., strong acids, ammonium sulfate).The Kjeldahl assay is a quantitative method for nitrogen determination also in crude samples. The method is based on the fact that nearly all proteins contain approximately 16.5% nitrogen by weight, which gives a conversion factor between nitrogen and protein content of approximately 6. The sample must be free of interfering nitrogen containing compounds.UV-spectrometry measures the absorbance at 277–280 nm (tyrosine and tryptophan), which is an indirect measure of the protein concentration. In protein mixtures an average extinction coefficient must be used and the method is consequently semiquantitative if not used relative to a quantitative method such as Kjeldahl. Major interfering agents are detergents, nucleic acids, particulates and lipid droplets.
Lindane degradation by root epiphytic bacterium Achromobacter sp. strain A3 from Acorus calamus and characterization of associated proteins
Published in International Journal of Phytoremediation, 2019
Protein profile of lindane degrading bacterial strain was studied by culturing the bacteria in MSM under two different conditions: (a) in the presence of glucose as carbon source (without lindane) and (b) lindane as sole carbon source. The bacterial cells were harvested aseptically from culture by centrifugation (8000 × g, 10 min, 4 °C). Pellets were washed twice with sterile phosphate buffer saline (0.05 M, pH 6.8); subsequently, pellet lysis buffer (Singh and Singh 2014) was added and boiled for 10 min. The cell lysate was brought to room temperature and sonicated (nine pulses of 5 s followed by 5 s cooling) at amplitude of 9 Hz. Protein concentration of cell lysate was determined using Bradford method. Electrophoresis was performed on discontinuous polyacrylamide gel (stacking 5% and separating gel10%) with 50 µg protein/lane at 70 V (through stacking gel for 2 h) further progressively increasing voltage to 120 V (through resolving gel for 5 h). Coomassie Brilliant Blue G-250 was used as staining dye for proteins. Gel was visualized on gel doc. Polypeptide bands were eluted from SDS-PAGE gel using ammonium bicarbonate and 50% acetonitrile solvent (Singh and Singh 2014). MALDI-TOF MS/MS analysis was done at AIRF JNU, New Delhi (Afreen et al. 2017).
Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid
Published in Preparative Biochemistry & Biotechnology, 2023
Jianjun Wang, Luyao Wang, Chengli Yang, Yihui Zhu, Ziqian Chen, Guanya He, Kaishun Hu, Kaixuan Liu, Beibei Fang, Dali Li, Ruofu Shi
Escherichia coli BL21 (DE3) (pET-30a-GAD) was incubated in kanamycin (50 µg mL−1)-containing in LB medium (5 mL) for 12 h (200 rpm, 37 °C), and then transferred to LB medium (500 mL) containing lactose (0.2%, w/v) and kanamycin (50 µg mL−1) for cultured under the same conditions, then collected cells by centrifugation (5 min, 4000×g) at 4 °C and washed with water three times. The cultured cells were disrupted by ultrasonic processor for 1 h and taken cell disruption solution as a crude extract. The SDS-PAGE was used to assay the proteins of the E. coli cell. The SDS-PAGE using separated gel (12%, m/v) and concentrated gel (5%, m/v). Coomassie Brilliant Blue R-250 staining detects proteins.[37]
Phytoremediation through Bidens pilosa L., a nonhazardous approach for uranium remediation of contaminated water
Published in International Journal of Phytoremediation, 2019
Muhammad Imran, Shanglian Hu, Xuegang Luo, Ying Cao, Naseem Samo
For this analysis, 4 mL of 80% ethanol was added to an appropriate mass of plant for extraction. To 1 mL of extract, 5 mL of onion ketone was added, placed in a boiling water bath for extraction. After cooling, the extract was analyzed by colorimetry at 625 nm and the concentration calculated. The soluble protein content was measured by Coomassie brilliant blue staining method. For protein analysis an appropriate amount of sample was ground thoroughly and homogenized with 5 mL of distilled water or buffer, the pellet centrifuged, and 1 mL of supernatant was mixed to 5 mL of Coomassie blue reagent, the solution was shaken for 2 min and then determined colorimetrically at 595 nm. The experiment was repeated three times for both sugar and protein. (Yu et al. 2016).