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Inherited Differences in Alpha1-Antitrypsin
Published in Stephen D. Litwin, Genetic Determinants of Pulmonary Disease, 2020
Recent purification procedures have used ammonium sulfate precipitation, gel chromatography, and ion exchange chromatography on DEAE-cellulose [14-17] and QAE-Sephadex [14]. Some methods employ a preparative electrophoretic step [18] in combination with ethanol precipitation [17]. The method of Pannell et al. [17], which includes an affinity chromatography step on blue dextran-Sepharose for the removal of albumin [20], has proven particularly useful because albumin has been a very persistent contaminant. Laurell et al. [21] have described an isolation procedure that employs affinity chromatography on glutathione-Sepharose as the essential innovative step. This step is preceded by another affinity chromatography on immobilized kappa light chains of immunoglobulin. Both steps use the principle of thiol-disulfide interchange chromatography. In both systems alpha antitrypsin is preferentially bound to glutathione or the C-terminal cysteine of kappa chains; it is then selectively displaced by a mixture of 3-carboxy-4-nitrobenzene thiol and 5,5'-dithiobis-(2-nitrobenzoate). The protein isolated by this technique is similar in amino acid composition to preparations obtained by different methods as listed in Table 2. There is good general agreement between the amino acid composition of alpha1-antitrypsin obtained by different authors. These results indicate that the various methods for purification result in a similar product. The advantage of one method over the other is largely in yield and the amount of labor involved.
Bioprocess Parameters of Production of Cyanobacterial Exopolysaccharide
Published in Gokare A. Ravishankar, Ranga Rao Ambati, Handbook of Algal Technologies and Phytochemicals, 2019
Onkar Nath Tiwari, Sagnik Chakraborty, Indrama Devi, Abhijit Mondal, Biswanath Bhunia, Shen Boxiong
The peripheral ultrafiltration method is projected as a substitute to the traditional extraction of EPS applying ethanol precipitation (Charcosset 2006). The salting out method is studied by applying the diafiltration process to facilitate the substitution of the solvent via a new buffer (Charcosset 2006). The ultrafiltration approach significantly diminishes the generation of “sieve bar” and passes the suitable concentration of the biomolecules reasonably to the final edge of the filtration process. The efficacy of the membrane system is solely reliant on the following points, (i) the thickness and concentration of EPS; (ii) the orifice mass allocation along with the architecture of membrane, and (iii) the speed of the flow and the pressure of the transmembrane.
Human Esophageal Epithelial Cells: Immortalization and In Vitro Transformation
Published in George E. Milo, Bruce C. Casto, Charles F. Shuler, Transformation of Human Epithelial Cells: Molecular and Oncogenetic Mechanisms, 2017
Gary D. Stoner, Zenya Naito, George E. Milo
High molecular weight DNA was isolated from precrisis HE-457 and postcrisis HET-1A cell lines using Pronase-sodium dodecyl sulfate lysis. Following inorganic extraction and ethanol precipitation, the DNA samples were treated with RNase and additional inorganic extractions, and ethanol precipitations were performed. The quantity of DNA isolated was determined in a spectrophotometer at 260 nm, and its integrity by electrophoresis in a 0.8% agarose gel. DNA samples were digested with the restriction endonu-clease HaeIII (Promega, Madison, WI), electrophoresed in a horizontal 0.8% agarose gel (10 μg DNA per lane), and transferred to a nylon membrane. The blots were hybridized under stringent conditions (50% formamide-0.75 M NaCl-0.075 M sodium citrate, 42°C) to a 32P-labeled DNA probe. The probe, designated pYNH24 (D2S44), was derived from a human genomic library.36,37 pYNH24 recognizes a single locus, hybridizes to human-specific repeated DNA fragments, and has been used for determination of a genotype specific for a single individual.36,37 The blots were washed to a final stringency in 0.1% sodium citrate-0.1% sodium dodecyl sulfate solution at 50°C. They were then exposed to film at -70°C with intensifying screens.
Pinocembrin polymeric micellar drug delivery system: preparation, characterisation and anti-hyperuricemic activity evaluation
Published in Journal of Microencapsulation, 2022
Wanjing Rong, Xinyi Shen, Michael Adu-Frimpong, Qing He, Jian Zhang, Xiaoxiao Li, Xiaoli Xia, Feng Shi, Xia Cao, Hao Ji, Elmurat Toreniyazov, Qilong Wang, Jiangnan Yu, Ximing Xu
Serum samples were treated by the ethanol precipitation method (Yang et al. 2019). The UA standard solutions of varied concentrations (1, 2.5, 5, 15 and 20μg/mL) were put into the blank serum (100μL). Chromatographic methanol (900µL) was added to precipitate proteins, vortically swirled (1min), before centrifugation for 10min at 10000rpm. After absorbing 400µL of supernatant into clean EP tubes, we added 0.2% acetic acid-water (400µL), vortex and mixed well, prior to filtering with organic membrane (0.22µm) and injection of filtrate based on chromatographic method described in Section 2.7.1. In-vivo standard curve of UA was constructed through linear regression method (y=32303x+6326.2, n=3, R2 = 0.9971), where the abscissa was the serum concentration of UA (C), and the ordinate was the peak area (A). The difference was within satisfactory limits after verification of the repeatability and accuracy of standard drugs.
Development of potent and effective synthetic SARS-CoV-2 neutralizing nanobodies
Published in mAbs, 2021
Maxwell A. Stefan, Yooli K. Light, Jennifer L. Schwedler, Peter R. McIlroy, Colleen M. Courtney, Edwin A. Saada, Christine E. Thatcher, Ashlee M. Phillips, Feliza A. Bourguet, Catherine M. Mageeney, Summer A. McCloy, Nicole M. Collette, Oscar A. Negrete, Joseph S. Schoeniger, Dina R. Weilhammer, Brooke Harmon
Library was assembled as follows. The linear library DNA fragment was amplified by PCR. PCR reaction was divided into a 384-well format to minimize potential amplification bias. Five cycles of PCR were performed (Step 1: 98°C, 3 minutes, 1 cycle; Step 2: 98°C, 10 seconds; 68°C, 10 seconds; 72°C, 15 seconds, 5 cycles; Step 3: 72°C, 10 minutes, 1 cycle; Step 4: 12°C, hold). Amplified DNA was pooled and purified by Monarch Nucleic Acid Purification Kit (NEB, T1020S). The library was digested with 5 U/μg DNA SfiI at 50°C for 16 hours. Digest reactions were again column purified as previously mentioned. Ligations were set up as follows. pADL20c (205 μg) previously digested with BglI and treated with rSAP was added to a 40 mL reaction containing the linear digested library (42 μg) at a vector to insert ratio of 1:2, 1 mM ATP and 1,100,000 units of T4 Ligase (Antibody Design Laboratories, PD0109) . The reaction was allowed to proceed at 16°C for 16 hours, then at 37°C for 1 hour, and lastly was heat inactivated at 70°C for 20 minutes. DNA was purified and concentrated by a modified ethanol precipitation protocol utilizing tRNA carrier at 15 μg/mL.
Extracellular microRNAs in blood differentiate between ischaemic and haemorrhagic stroke subtypes
Published in Journal of Extracellular Vesicles, 2020
M. Yashar S. Kalani, Eric Alsop, Bessie Meechoovet, Taylor Beecroft, Komal Agrawal, Timothy G. Whitsett, Matthew J. Huentelman, Robert F. Spetzler, Peter Nakaji, Seungchan Kim, Kendall Van Keuren-Jensen
Library construction for sequencing was performed as previously described [17,33]. Briefly, we used the TruSeq small RNA sample preparation kit (RS-200-0048; Illumina) with half the reaction volume and reagents. RNA adaptors (3′ and 5ʹ) are specifically ligated to small RNAs, and the resulting construct is reverse transcribed. The construct is then enriched by PCR (16 cycles) during which a unique barcode was added to the sequence. The libraries were then run on a 6% TBE Gel for 55 min at 140 V and the product between 140 to 160 bp were excised from the gels. These gel pieces were fractured into smaller pieces and allowed to incubate on a rotator overnight in water. An ethanol precipitation was performed to precipitate the RNA and the resulting pellet of RNA was resuspended in 11 ul of ultra-pure water. Samples were quantified with the Agilent High Sensitivity DNA Kit (5067–4626; Agilent). The peak for the sample was integrated from 120 bp to 160 bp to get the pMolarity. Samples with similar pMolarity were grouped on the same lane and pooled together to create a total of 8 unique pools containing 15 samples with different barcodes. These pools were then quantified with the Agilent High Sensitivity DNA Kit to get the final pMolarity for the pool. The pools were then denatured and clustered on a single read Illumina V3 flowcell (GD-401-3001; Illumina). Illumina HiSeq 2500 next-generation sequencing platform was used for all sequencing experiments. All samples were sequenced to approximately the same depth/number of reads so that quantitative comparisons can be made between samples.