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Assay Requirements for Cell Culture Process Development
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
Mary B. Sliwkowski, Edward T. Cox
Protein Stains. For analysis of purified proteins or for detecting total protein following electrophoretic separation, several procedures are available for direct staining of gels. Silver stains provide the highest level of sensitivity, ~1 ng. Merril (53) recently reviewed the many different silver-staining procedures which have been developed. Some protocols produce different colored bands for many proteins (54). This is useful for identifying uniquely colored proteins in complex patterns, such as seen with 2D gels, but the intensity variations make quantitation more difficult. We find the Morrissey (55) method to give the best combination of speed, sensitivity, and reliability. Response to silver staining varies among individual proteins, and different applications may require different protocols.
Attenuation of streptozotocin induced high fat diet exacerbated dyslipidemia mediated hepatic and aortic injuries in male pigs by camel milk
Published in Egyptian Journal of Basic and Applied Sciences, 2023
Hadiza Bello Rilwan, Sunday Samuel Adebisi, James Abrak Timbuak, Sunday Blessing Oladele, Aliyu Muhammad, Wusa Makena, Adamu Abubakar Sadeeq
Photomicrograph section of liver tissue from the control group NC showed normal hepatocytes, hepatic portal vein present, hepatic artery, bile duct, and lymphatic space visible conspicuously between the hepatocyte and the hepatic portal artery (Figure 5a), a histological segment of D-HFD pigs with fat hepatocellular vacuoles of varying sizes, ballooning degeneration, and necrosis (Figure 4b). A photomicrograph of pigs in the D-HFD + 250 CM group demonstrated regular progressive repair of hepatocytes, minor inflammation, ballooning degeneration, and hepatocyte necrosis (Figure 4c). Sections from the D-HFD + 500 CM group showed standard portal triad, remarkable hepatocyte restoration, and mild ballooning degeneration of some hepatocytes (Figure 4d). In contrast, D-HFD + MET showed gradual hepatocyte restoration and mild inflammation, ballooning degeneration, and hepatocyte necrosis (Figure 4e). For Gordon and Sweet Silver Stain, the liver of the NC sections showed normal distribution of reticular fibers in the parenchyma reticular fiber (Figure 5a). The hepatic distribution of diabetic/atherosclerotic pigs revealed substantial (chronic) reticular fiber loss (Figure 5b). Treatment D-HFD + 250 CM group demonstrated minimal reticular fiber repair (Figure 5c), D-HFD + 500 cm group demonstrated marked reticular fiber repair (Figure 5d), while D-HFD + MET group showed marked reticular fiber repair (Figure 5e).
Sericin from mulberry and non-mulberry silk using chemical-free degumming
Published in The Journal of The Textile Institute, 2022
Monalisa Kalita, Benjamin J. Allardyce, Kamatchi Sankaranarayanan, Dipali Devi, Rangam Rajkhowa
The protein concentration of the sericin solution obtained from the Ahiba degummed cocoons was determined by Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as the standard . The Bradford’s assay kit can estimate samples having protein concentrations between 0.2 and 1.4 mg/ml. The protein amount was estimated by measuring the absorbance at 595 nm using Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific). The amount of protein present in B. mori, A. assamensis and P. ricini was estimated to be 1.03 mg/ml, 0.88 mg/ml and 0.92 mg/ml respectively. The molecular weight profile of sericin obtained was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method (Laemmli, 1970). Briefly, hand cast 8% polyacrylamide gels were prepared and 1X Tris/glycine/SDS running buffer (pH = 8.3) was used for electrophoresis at a constant 90 V and run time of 120 mins. The gels were stained with silver stain following the sequential phases of protein fixation, sensitization, silver impregnation and image development.
Toxicoproteomic assessment of liver responses to acute pyrrolizidine alkaloid intoxication in rats
Published in Journal of Environmental Science and Health, Part C, 2018
Yan-Hong Li, William Chi-Shing Tai, Imran Khan, Cheng Lu, Yao Lu, Wing-Yan Wong, Wood-Yee Chan, Wen-Luan Wendy Hsiao, Ge Lin
Proteomic studies were performed on the liver specimens of three rats per group at 24 hr dosed with 140 mg/kg RTS, or saline. The protein samples of individual rat liver tissues were extracted by homogenizing with 2D protein extraction buffer (9 M urea, 65 mM DTT, and 2% CHAPS) and then centrifuged at 100,000 × g for 1 hr at 10°C. The supernatants were collected and protein concentration was determined by 2D Quant Kit (GE Healthcare, Fairfield, CT, USA). Individual protein sample (120 µg) dissolved in 250 µl rehydration buffer (8 M urea, 24% CHAPS, 2% DTT, 0.5% IPG ampholytes, and 1% bromophenol blue) was loaded on IPG strips (4–7 linear pH gradients, 13 cm long). Isoelectric focusing (IEF) was then carried out in an EttanIPGphor 3 IEF System (GE Healthcare) at 80,000 V/h. The resultant strips were equilibrated in SDS equilibration buffer (6 M urea, 75 mM Tris-HCl buffer, pH 8.8, 30% (v/v) glycerol, and 2% SDS) and then subjected to 12.5% SDS polyacrylamide gel separation. Protein spots were visualized in silver stain sensitizing solution.