Separation of Free and Bound cAMP in Mycobacteria
在分枝杆菌中分离游离和结合的cAMP
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1879 浏览次数: 8054
评审: Valentine V TrotterDarrell CockburnJoana Antunes
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
Mycobacterial genomes encode a plethora of genes that are involved in the synthesis, utilization and degradation of cAMP. The genome of M. tuberculosis H37Rv, for example, encodes 16 adenylyl cyclases and 10 genes harbouring the cyclic nucleotide-binding (CNB) domain (Shenoy and Visweswariah, 2006). Cyclic AMP is efficiently secreted by mycobacteria, and cytosolic as well as extracellular levels of cAMP can reach hundreds of micromolar. We have recently reported that an abundantly expressed universal stress protein (USP; Rv1636 in M. tuberculosis H37Rv and MSMEG_3811 in M. smegmatis, respectively) binds cAMP (Banerjee et al., 2015). Given the number of cAMP-binding proteins present in mycobacteria, it is expected that a significant fraction of intracellular cAMP may be bound to protein. The methods typically employed to measure cAMP are radioimmunoassay (RIA) and ELISA. However, these procedures include prior acidification of samples that would dissociate cAMP ‘bound’ to protein, and therefore represent the ‘total’ cAMP present in the sample. In this protocol, we describe a method to separate the fraction of cAMP ‘bound’ to protein from what is ‘free’ or not associated with protein. This is performed by subjecting the cytosolic fraction or the culture supernatant to filtration through a membrane with a 3 kDa cut-off. Only ‘free’ cAMP is able to pass through the membrane. Therefore, cAMP concentrations in the filtrate represent the ‘free’ cAMP in the sample. Cyclic AMP levels in the original cytosolic fraction or the culture supernatant represent the ‘total’ cAMP concentration. Subtracting the ‘free’ from the ‘total’ provides the amount of cAMP bound to protein.
Keywords: CAMP (营)
CAMP-binding protein (cAMP结合蛋白)
Mycobacterium (结核分枝杆菌)
Universal stress protein (普遍的应激蛋白)
Materials and Reagents
- 15 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339650 )
- 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-175-C )
- T25 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 707003 )
- T75 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 708003 )
- Amicon Ultra-0.5 ml 3-kDa centrifugal filter with Ultracel-3 membrane (Merck Millipore Corporation, catalog number: UFC500324 )
- 50 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339652 )
- Glass test tubes for growing 5 and 10 ml cultures.
- 96 well polystyrene plates (Sigma-Aldrich, product number: M2936 )
- 0.5 mm diameter glass beads (Bio Spec Products Inc, catalog number: 11079105 )
- 2 ml polypropylene bead beating vials (Bio Spec Products Inc, catalog number: 10831 )
- Mycobacterium smegmatis mc2 155 (ATCC, catalog number: 700084 )
- Mycobacterium bovis BCG (ATCC, catalog number: 35734 )
- Middlebrook 7H9 medium (BD Biosciences, catalog number: 271310 )
- Glycerol (Thermo Fisher Scientific, catalog number: 17904 )
- Magnesium sulfate (Merck Millipore Corporation, catalog number: 105886 )
- Tween 80 (Sigma-Aldrich, product number: P 4780 )
- 7H9-Glycerol-Tween 80 medium
- Oleic acid/albumin/dextrose/catalase (OADC) growth supplement (BD Biosciences, catalog number: 211886 )
- Bradford reagent (Sigma-Aldrich, catalog number: B6916 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
- Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144SI-212 )
- cAMP ELISA kit (Enzo Lifesciences, catalog number: ADI-900-067 )
- cAMP Direct Immunoassay Kit (Abcam plc, catalog number: ab65355 )
- Cyclic AMP Competitive ELISA Kit (Thermo Fisher Scientific, catalog number: EMSCAMPL )
- Sodium chloride (Merck Millipore Corporation, catalog number: 1064040500 )
- Potassium chloride (Merck Millipore Corporation, catalog number: 1049360500 )
- Di-Sodium hydrogen phosphate (Merck Millipore Corporation, catalog number: 1065860500 )
- Potassium dihydrogen phosphate (Merck Millipore Corporation, catalog number: 1048730250 )
- Tris(hydroxymethyl)aminomethane (Merck Millipore Corporation, catalog number: 1083820500 )
- 2-Mercaptoethanol (Sigma-Aldrich, product number: M6250 )
- Phenylmethylsulfonyl fluoride (Sigma-Aldrich, product number: PMSF-RO ROCHE)
- Phosphate buffered saline (see Recipes)
- Lysis buffer (see Recipes)
Equipment
Note: Biosafety level 2 (BSL 2) is required for culturing M. bovis BCG
- 37 °C incubator (humidified)/shaker (N-BIOTEK, model: NB-203QR /Eppendorf, New BrunswickTM Innova®, model: 40 )
- Spectrophotometer (Eppendorf, BioPhotometer®, model: D30 )
- Refrigerated centrifuge (Thermo Fisher Scientific, HeraeusTM MegafugeTM, model: 16R ) equipped with TX-400 Swinging Bucket Rotor (Thermo Fisher Scientific, model: TX-400 ) and adapter (number 20 & 23)
- Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17R )
- Vortexer (Bio-rad, model: BR-2000 )
- Plate reader (reads in the visible spectrum, including 405 and 595 nm) (Tecan, Infinite®, model: m200 PRO )
- Bead beater (BioSpec Products, model: Mini-Beadbeater-16 )
- Heating block (heats to 95 °C) (NeoLab)
- Ultra low temperature freezer (Panasonic, model: MDF-U33V-PE )
Software
- GraphPad Prism 5
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Banerjee, A. and Visweswariah, S. S. (2016). Separation of Free and Bound cAMP in Mycobacteria. Bio-protocol 6(14): e1879. DOI: 10.21769/BioProtoc.1879.
- Banerjee, A., Adolph, R. S., Gopalakrishnapai, J., Kleinboelting, S., Emmerich, C., Steegborn, C. and Visweswariah, S. S. (2015). A universal stress protein (USP) in mycobacteria binds cAMP. J Biol Chem 290(20): 12731-12743.
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分类
微生物学 > 微生物生理学
微生物学 > 微生物信号传导 > 第二信使
生物化学 > 其它化合物 > 环磷酸腺苷（cAMP）
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