Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration
蛋白质提取、酸性磷酸酶活性分析以及可溶性蛋白浓度的测定
发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.889 浏览次数: 22966
评审: Tie Liu
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参见作者原研究论文
The authors used this protocol in:
Nov 2012
Abstract
Acid phosphatases (APases) catalyze the hydrolysis of inorganic phosphate (Pi) from a broad range of Pi-monoesters with an acidic pH optimum. The liberated Pi is reassimilated into cellular metabolism via mitochondrial or chloroplastic ATP synthases of respiration or photosynthesis, respectively. Eukaryotic APases exist as a wide variety of tissue- and/or cellular compartment-specific isozymes that display marked differences in their physical and kinetic properties. Increases in intracellular (vacuolar) and secreted APase activities are useful biochemical markers of plant nutritional Pi deficiency. The protocols for protein extraction, APase activity determination and measurement of soluble protein concentration from plant tissues or cell suspension cultures are presented.
Materials and Reagents
- Plant tissues
- Sodium acetate (Bioshop, catalog number: SAA305 )
- EDTA (Bioshop, catalog number: EDT001 )
- Dithiothreitol (DTT) (Bioshop, catalog number: DTT 002 )
- Phenylmethyl sulfonyl fluoride (PMSF) (G-Biosciences, catalog number: 786-0555 )
- Thiourea (Sigma-Aldrich, catalog number: T-7875 )
- Polyvinyl (polypyrrodlidone) (PVPP) (Sigma-Aldrich, catalog number: P-6755 )
- β-nicotinamide adenine dinucleotide reduced form (NADH) (Bioshop, catalog number: NAD002 )
- Phosphoelnolpyryvate (PEP) (Biovectra, catalog number: 2552 )
- MgCl2 ((Biolynx, catalog number: 18641 )
- Rabbit muscle lactate dehydrogenase (LDH) (Sigma-Aldrich, catalog number: L-2500 )
- para-nitrophenyl-phosphate (pNPP, phosphatase substrate) (Sigma-Aldrich, catalog number: P-4744 )
- Coomassie Brilliant blue G-250 (Serva, catalog number: 35050 C.!.42655 )
- Bovine gamma globulin (BGG) (2.0 mg/ml) (Thermo Fisher Scientific, catalog number: 23212 )
- NaOH
- Extraction buffer (EB) (see Recipes)
- Bradford working solution (see Recipes)
- Acid phosphatase enzyme assay mix #1 (see Recipes)
- Acid phosphatase enzyme assay mix #2 (see Recipes)
- Bradford assay stock (see Recipes)
Equipment
- Whatman #1 filter paper
- 1.5 ml microfuge tubes
- 10 and 25 μl Hamilton syringes
- Pipetor
- Small mortar and pestle
- Eppendorf microfuge
- 96 well polystyrene microtitre plate (flat bottom)
- A computer supported microplate spectrophotometer (Spectromax Plus, Molecular Devices, Sunnyvale)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Knowles, V. and Plaxton, W. (2013). Protein Extraction, Acid Phosphatase Activity Assays, and Determination of Soluble Protein Concentration. Bio-protocol 3(17): e889. DOI: 10.21769/BioProtoc.889.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
植物科学 > 植物生物化学 > 蛋白质
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