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Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
The most common method for quantifying proteins is the bicinchoninic acid (BCA) method, which, like the Lowry method, depends on the reaction being under alkaline conditions of Cu2+ with specific amino acid residues converting to the Cu+ form. This is detectable as a colorimetric change from green to purple (562 nm); however, this suffers from sensitivity to the presence of reducing agents. This method is also dependent on the amino acid sequence of the assayed proteins themselves, as this conversion varies based on the presence of cysteine, cystine, tyrosine, or tryptophan groups. As these groups are in low abundance in collagen, this may not be suitable for protein solutions of cartilage that contain abundant collagen. An alternate technique, the Bradford assay, is based on the binding of Coomassie blue and absorbance shift to 595 nm of the blue dye. Preferentially, this binding is to the side groups on the arginine, phenylalanine, proline, and tryptophan residues, but this assay suffers from sensitivity to detergents (such as SDS). Other techniques are also available for protein quantification; however, the ease of use of the BCA and Bradford techniques has made them extremely common. In practical use, these limitations in detection are rarely important, as most quantification is relative. Commonly, quantification is between sample treatment groups, which are rarely different enough in total protein profile to cause problems using these techniques. As with any protein quantification technique, the preparation of an appropriate standard curve within the linear range of the particular assay is paramount. Generally, bovine serum album (BSA) will give satisfactory results as a protein standard, especially where determination of relative protein quantification is more important, for example, to standardize gel loading on a Western blot.
Synthesis and Engineering of Polymeric Latex Particles for Hemodialysis Part II—An Experimental Study
Published in Wolfgang Sigmund, Hassan El-Shall, Dinesh O. Shah, Brij M. Moudgil, Particulate Systems in Nano- and Biotechnologies, 2008
S. Kim, H. El-Shall, R. Partch, T. Morey, B. Koopman
Protein adsorption on latex particles was performed with bovine serum albumin (BSA) as a standard model protein and β2-microglobulin (β2M) as a target protein in two types of buffer solution: phosphate buffer (PB) and phosphate buffered saline (PBS). 0.345 g of sodium phosphate monobasic (NaH2PO4·H2O) was dissolved in 500 mL DI water to make 5 mM PB solution. 0.345 grams of sodium phosphate monobasic (NaH2PO4·H2O) and 4.178 g (143 mM) sodium chloride (NaCl) were dissolved in 500 mL DI water to make the PBS solution. The synthesized latex particles were diluted with each buffer to be 0.5% (w/w) solid content and adjusted to the desired pH values such as 3.2, 4.8 and 7.4, and mixed with a selected protein, where BSA concentration were 0.05, 0.1, 0.3, 0.5 and 0.7 mg/mL and β2M were 0.015, 0.030, 0.045 and 0.060 mg/mL. Each mixture was gently rotated in the incubator at 37°C for 12 h. Thereafter, the latex-protein mixture was centrifuged at 13,000 rpm for 15 min. The amount of protein adsorbed was determined by quantifying the free protein in the supernatant after centrifugation using the bicinchoninic acid (BCA) assay method (Lowry et al., 1951; Smith et al., 1985; Wiechelman, Braun, and Fitzpatrick, 1988; Brown, Jarvis, and Hyland, 1989; Baptista et al., 2003). The BCA assay kit consists of Reagent A containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 N NaOH with pH = 11.25 and Reagent B containing 4% (w/v) copper (II) sulfate pentahydrate. The BCA working reagent was prepared by mixing 50 parts Reagent A with 1 part Reagent B. 100 μL of protein supernatant was then mixed with 2 ml BCA working reagent in UV cuvette. Incubation was allowed for color development at room temperature for 2 h. The absorbance on a UV-VIS spectrophotometer (Perkin-Elmer Lambda 800, USA) at 562 nm was read. The unknown concentration of sample protein was determined using a standard curve, which was established with already-known concentrations of a protein. The adsorbed amount per unit surface area was determined by the mass balance of the protein.
Optimized protocol for the biocompatibility testing of compression stockings and similar products with close skin contact in vitro
Published in The Journal of The Textile Institute, 2018
Cornelia Wiegand, Tanja Hansen, Johanna Köhnlein, Ines Exner, Marlen Damisch-Pohl, Peter Schott, Ulrike Krühner-Wiesenberger, Uta-Christina Hipler, Ernst Pohlen
After the respective incubation times, cell culture medium was removed completely and cells were washed twice with PBS. Cells were lysed with 0.1% Triton-X 100 in PBS at 90 °C for 10 min. After cooling, the plates were stored at −20 °C until testing. Total protein content was determined by the Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA U.S.A.) based on the biuret reaction. The method combines the reduction of Cu2+ to Cu+ by protein in an alkaline solution with the colorimetric detection of Cu+ using bicinchoninic acid (BCA). 200μL of the BCA reagent were added to 25μL lysate followed by 30 min incubation at 60 °C. Absorbance was measured at 580 nm using a microplate photometer FLUOstar Galaxy (BMG Labtech, Ortenberg, Germany). Protein concentrations were calculated on the basis of a BSA (bovine serum albumin) standard curve provided in the Pierce BCA Protein Assay. The standard curve was prepared as recommended by the manufacturer.