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Inherited Differences in Alpha1-Antitrypsin
Published in Stephen D. Litwin, Genetic Determinants of Pulmonary Disease, 2020
Alpha1-antitrypsin was first described by Schultze et al. [10] as alpha1-3,5-glycoprotein. In a subsequent report it was identified as a trypsin inhibitor and renamed alpha1-antitrypsin [11]. Its purification was achieved by a series of precipitation steps and a final preparative zone electrophoresis on Polyvinylchloride. Partial purification and characterization was also reported by Moll et al, [12] and Bundy and Mehl [13] using ammonium sulfate precipitation and ion exchange chromatography.
Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
Supernates from the wells containing hybridomas were assayed for anti-PRL receptor activity using as criteria the inhibition of 125I-hGH binding to rabbit mammary microsomes and the ability to immunoprecipitate 125I-hGH-receptor complexes. Labeled hGH was used rather than labeled PRL, since both hormones are lactogenic and bind to the PRL receptor; in addition, since PRL was used for the affinity purification, the possibility of selecting hybrids producing anti-PRL serum was minimized. Supernates (20 to 50 μℓ) were added to the standard PRL binding assay reaction mixture 1 hr before the addition of 125I-hGH. Positive hybridomas were cloned twice by the limiting dilution or the soft agar method.73 The cloned hybridomas were grown in medium and injected to BALB/c mice primed with Pristane. Antibodies were harvested as ascites fluid. Antibodies were purified by ammonium sulfate precipitation and further purified on protein A-agarose.74
General Introduction
Published in David N. Brindley, John R. Sabine, Phosphatidate Phosphohydrolase, 2017
The molecular weight distributions of phosphatidate phosphohydrolase from the cytosolic fraction of rat liver or from microsomal fractions that had been sonicated or treated with Tween 20 have been characterized.55 The method employed ammonium sulfate precipitation followed by chromatography on Bio-Gel A-5 m. The activity was detected by using microsomal membranes containing phosphatidate, phosphatidate dispersed by sonication with microsomal lipids, or in solvent-disrupted microsomes. The results demonstrate that the molecular weight profile of the phosphohydrolase activity is very dependent on the physical state of the substrate (see also Section II.B) and that the phosphohydrolase activity may form into aggregates. Similar conclusions are described for the phosphohydrolase activity in lung. The elution profile of phosphohydrolase activity from gel filtration columns can also be modified by incubating the enzyme preparation with phospholipids.54
Effects of the Cobalt-60 gamma radiation on Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase
Published in International Journal of Radiation Biology, 2022
Abdelghani Iddar, Mohammed El Mzibri, Adnane Moutaouakkil
The GAPDH was purified from normal culture of P. pastoris by selective precipitation with ammonium sulfate flowed by chromatography on Cibacron blue CL-6B. Table 1 summarizes a representative purification of P. pastoris GAPDH. Specific activity value of 52.3 ± 1.2 U/mg of proteins was obtained for the purified enzyme, with yield of ca. 29.5%. The protein was purified ca. 43.2-fold after the Cibacron blue chromatography. The ammonium sulfate precipitation represented a large contribution to the overall purification (the purification factor was 15.4). The enzyme activity was eluted from the dye-ligand affinity chromatography column as a single symmetrical peak. SDS-PAGE analysis of the different fractions obtained during the purification procedure showed a progressive enrichment of a 35-kDa protein (Figure 1(A)). The apparent subunit molecular mass was determined by measuring relative motilities and comparing with pre-stained SDS-PAGE molecular mass protein standards. Only this protein band, which corresponds to the GAPDH subunit, was observed in the electrophoretically homogeneous final enzyme preparation (Figure 1(A)). Rabbit polyclonal antibodies have been produced using purified P. pastoris GAPDH as immunogen. These antibodies selectively reacted by the immunoblotting procedure with a single immunoreactive band in crude extract and purification steps. Figure 1(B) shows that the relative molecular mass of the detected protein (35 kDa) is the expected one for the GAPDH monomer.
Some indazoles reduced the activity of human serum paraoxonase 1, an antioxidant enzyme: in vitro inhibition and molecular modeling studies
Published in Archives of Physiology and Biochemistry, 2019
Zuhal Alım, Deryanur Kılıç, Yeliz Demir
Triton X-100 treated human serum (20 ml) was subjected to ammonium sulfate precipitation in the range of 60–80%. After centrifugation (at 1500 g for 20 min), precipitate was collected and redissolved in 100 mM Na-phosphate buffer (pH 7.0). The enzyme solution was dialyzed against 1 mM sodium phosphate buffer (pH 7.0) for about 2 h. Then, the enzyme solution was loaded onto the DEAE-Sephadex A50 anion exchange column previously equilibrated with the 100 mM Na-phosphate buffer (pH 7.0). The column was washed with a 100 mM Na-phosphate buffer to remove any other impurities. A linear gradient of 0–1.5 M NaCl (100 ml) was used to elute the enzyme. The activity measurements were made at 412 nm in all the elutions, and the active fractions were pooled. After anion exchange chromatography, active enzyme solution was loaded onto the gel filtration column (Sephadex G-100) equilibrated with a 100 mM Na-phosphate buffer (pH 7.0). Both the qualitative protein identification (280 nm) and enzyme activity (412 nm) were monitored in the eluates. The tubes observed in the enzyme activity were combined for other kinetic studies. All purification procedures were made at 4 °C. These processes were performed as in our previous studies (Isgor and Beydemir 2010, Alim and Beydemir 2016). Quantitative protein assay was performed using the Bradford method (Bradford 1976) and the enzyme purity was controlled according to Laemmli’s procedure (Laemmli 1970, Demir and Beydemir 2015).
A computational method for predicting the aggregation propensity of IgG1 and IgG4(P) mAbs in common storage buffers
Published in mAbs, 2022
James T. Heads, Sebastian Kelm, Kerry Tyson, Alastair D. G. Lawson
To establish the native state aggregation propensity of a mAb, the preferred approach is to concentrate the sample, then apply various analytical techniques to detect the formation of aggregates. This methodology requires large amounts of purified sample (up to 200 mg), which precludes its use early in the antibody drug discovery phase where sample availability is limited. Other methods to determine aggregation propensity that require smaller quantities of sample (<0.5 mg) include ammonium sulfate-induced precipitation,9 affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS)10,11 and the polyethylene glycol (PEG)-induced protein precipitation assay.12–15 We selected PEG precipitation to study native state aggregation because ammonium sulfate precipitation requires the addition of high salt concentrations, which can perturb native electrostatic interactions between protein molecules. Suppression of electrostatic interactions could bias results toward a hydrophobicity-driven predictive model. AC-SINS is reported to be an effective measure of protein–protein interactions and is compatible with very dilute solutions (1–100 µg/mL). However, the assay requires the test mAb to be immobilized, therefore a substantial portion of the molecule is obscured limiting interactions that would otherwise occur in solution. A study examining the developability of 137 clinical stage mAbs suggested that this assay was likely to be a measure of self-interaction and varying levels of cross interactions with the nanoparticle complexes.16 This observation could explain why samples sharing similar AC-SINS scores were shown to have remarkably different solubility values.17,18 These reports suggested that the AC-SINS assay was not a suitable method for detailed analysis of aggregation propensity.