Useful Principles for the Crystallization of Proteins
Hartmut Michel in Crystallization of Membrane Proteins, 1991
The most common procedure for inducing proteins to separate from solution and enter the solid state is to gradually increase the level of saturation of a salt. Traditionally the salt has been ammonium sulfate, but others (see section on precipitants) are also in wide use. Usually, the protein separates as a precipitate, but with appropriate care, increase in salt concentration can be used to grow protein crystals. At this writing, in fact, this approach has probably yielded more varieties of protein crystals than any other.
American Ginseng Farm in the State of Wisconsin
Joseph P. Hou in The Healing Power of Ginseng, 2019
Nitrogen needs a range from 20 to 60 lb/acre, depending on soil organic matter level. (However, some growers have been known to use considerably more.) Growers have tended to use lower-salt fertilizers, such as ammonium sulfate, potassium sulfate, and potassium-magnesium sulfate. Although secondary and/or micronutrients are often involved in fertilization programs, little research has been conducted to confirm responsiveness.
Phosphatidylinositol and inositolphosphatide metabolism in hypertrophied rat heart
H. Saito, Y. Yamori, M. Minami, S.H. Parvez in New Advances in SHR Research –, 2020
Briefly, the cytosolic fraction was precipitated by adding ammonium sulfate (10-60%). The activity was observed in a 20-40%-ammonium sulfate fraction. This fraction was dialyzed against 10 mM Tris-malate buffer (pH 7.0), and applied to a DEAE cellulose column (Whatman, DE 52). A sodium chloride linear gradient system (0-400 mM) in 10 mM Tris-malate buffer (pH 7.0) was used to elute IP3 kinase.
Exosome mimetics derived from bone marrow mesenchymal stem cells deliver doxorubicin to osteosarcoma in vitro and in vivo
Published in Drug Delivery, 2022
Jinkui Wang, Mujie Li, Liming Jin, Peng Guo, Zhaoxia Zhang, Chenghao Zhanghuang, Xiaojun Tan, Tao Mi, Jiayan Liu, Xin Wu, Guanghui Wei, Dawei He
Adherent cells were isolated by scraping, and the collected BMSCs were re-suspended in phosphate-buffered saline (PBS) at a concentration of 5 × 106 cells/mL. The Extruder (Avanti Mini-Extruder) was used for two sequential extrusions through a 10-μm and 5-μm polycarbonate membrane filter (Whatman), as in the previous article (Jang et al., 2013). Then super centrifuged at 100,000g for 70 min and re-suspend in 240 mM ammonium sulfate solution. The monodisperse nanoscale EMs were then prepared using a miniature extruder (Avanti Mini-Extruder) through a 1-μm polycarbonate membrane filter (Whatman). Then slide-A-Lyzer Nutritional Cassette (MWCO 20 kDa) was used overnight at room temperature at 2 L PBS (pH7.4) to remove ammonium sulfate outside EMs and form an ammonium sulfate concentration gradient. Then doxorubicin was added to EMs with a concentration of 1010 particles/mL at a final concentration of 0.2–1 mg/mL and incubated at room temperature for 6 h to promote the loading of doxorubicin into EMs. The preparation method of ammonium sulfate was described in previous literature (Guo et al., 2021). A doxorubicin-encapsulating EM (EM-Dox) solution was injected into slide-A-Lyzer Nutritional Cassette (MWCO 20 kDa) and dialyzed in PBS (pH7.4) overnight at room temperature to remove free doxorubicin. We used the same method to prepare the blank EMs using a miniature extruder (Avanti mini-extruder) through a 10-μm, 5-μm, and 1-μm polycarbonate membrane filter (Whatman) for three sequential extrusions.
Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2022
Jina Ryu, Sung Jae Yang, Boram Son, Haein Lee, Jongmin Lee, Jinmyoung Joo, Hee Ho Park, Tai Hyun Park
To optimize amount of MMP-2 for LK cleavage, recombinant ASNase-LK-30Kc19 proteins were incubated with 0.05–0.2 μg of MMP-2 (Calbiochem) at 37 °C for 6 h. Direct nesslerization method was performed to measure the amounts of ammonia formed [40,41]. Briefly, L-Asn was added to the pre-incubated mixture of ASNase-LK-30Kc19 and 0.1 μg MMP-2, and then incubated at 37 °C for 30 min. The reaction was stopped by adding 1.5 M Trichloroacetic acid, and then Nessler’s reagent (Sigma, St. Louis, MO, USA) was added. We prepared 1 mM ammonium sulphate ((NH4)2SO4) and used it in the standard range of 0–100 nmol. The blank was used to filter out the artefacts (△A450nm Standard=△A450nm Standard−△A450nm Blank). Coloured compound from the reaction of Nessler’s reagent and ammonia was formed, and microplate reader (Thermo Scientific) was used to measure absorbance at 450 nm.
Affinity enrichment for mass spectrometry: improving the yield of low abundance biomarkers
Published in Expert Review of Proteomics, 2018
Brianna Kim, Robyn Araujo, Marissa Howard, Ruben Magni, Lance A. Liotta, Alessandra Luchini
Precipitation or ‘salting out’ of proteins is another common method for concentrating proteins from large volumes. Ammonium sulfate is a common reagent used for this purpose. Increasing the concentrations of ammonium sulfate elevates the ionic strength of the solution. The increase in ionic strength reduces the repulsion of same charges of protein molecules and reduces the solvation shell around proteins. Once these forces are sufficiently reduced the proteins will interact, aggregate, and precipitate. The precipitated proteins can be collected by centrifugation and be resolubilized in a smaller volume to achieve a concentration effect. A drawback of salting out is functional disruption of biologic activity when the protein is in an insoluble state. Moreover, the proteins may not return to solution phase upon resolubilization. Commercial kits exist to conduct salting-out using proprietary reagents that protects the biological activity of the proteins, and improve resolubilization [20].
Related Knowledge Centers
- Ammonium
- Ammonium Nitrate
- Fungicide
- Insecticide
- Ph
- Sulfur
- Nitrogen
- Salt
- Herbicide
- 2,4-Dichlorophenoxyacetic Acid