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Separation of Free and Bound cAMP in Mycobacteria
在分枝杆菌中分离游离和结合的cAMP   

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参见作者原研究论文

本实验方案简略版
The Journal of Biological Chemistry
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

  1. 15 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339650 )
  2. 1.5 ml microcentrifuge tubes (Corning, Axygen®, catalog number: MCT-175-C )
  3. T25 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 707003 )
  4. T75 polystyrene tissue culture treated flasks (Nest Biotechnology, catalog number: 708003 )
  5. Amicon Ultra-0.5 ml 3-kDa centrifugal filter with Ultracel-3 membrane (Merck Millipore Corporation, catalog number: UFC500324 )
  6. 50 ml polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339652 )
  7. Glass test tubes for growing 5 and 10 ml cultures.
  8. 96 well polystyrene plates (Sigma-Aldrich, product number: M2936 )
  9. 0.5 mm diameter glass beads (Bio Spec Products Inc, catalog number: 11079105 )
  10. 2 ml polypropylene bead beating vials (Bio Spec Products Inc, catalog number: 10831 )
  11. Mycobacterium smegmatis mc2 155 (ATCC, catalog number: 700084 )
  12. Mycobacterium bovis BCG (ATCC, catalog number: 35734 )
  13. Middlebrook 7H9 medium (BD Biosciences, catalog number: 271310 )
  14. Glycerol (Thermo Fisher Scientific, catalog number: 17904 )
  15. Magnesium sulfate (Merck Millipore Corporation, catalog number: 105886 )
  16. Tween 80 (Sigma-Aldrich, product number: P 4780 )
  17. 7H9-Glycerol-Tween 80 medium
  18. Oleic acid/albumin/dextrose/catalase (OADC) growth supplement (BD Biosciences, catalog number: 211886 )
  19. Bradford reagent (Sigma-Aldrich, catalog number: B6916 )
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
  21. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144SI-212 )
  22. cAMP ELISA kit (Enzo Lifesciences, catalog number: ADI-900-067 )
  23. cAMP Direct Immunoassay Kit (Abcam plc, catalog number: ab65355 )
  24. Cyclic AMP Competitive ELISA Kit (Thermo Fisher Scientific, catalog number: EMSCAMPL )
  25. Sodium chloride (Merck Millipore Corporation, catalog number: 1064040500 )
  26. Potassium chloride (Merck Millipore Corporation, catalog number: 1049360500 )
  27. Di-Sodium hydrogen phosphate (Merck Millipore Corporation, catalog number: 1065860500 )
  28. Potassium dihydrogen phosphate (Merck Millipore Corporation, catalog number: 1048730250 )
  29. Tris(hydroxymethyl)aminomethane (Merck Millipore Corporation, catalog number: 1083820500 )
  30. 2-Mercaptoethanol (Sigma-Aldrich, product number: M6250 )
  31. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, product number: PMSF-RO ROCHE)
  32. Phosphate buffered saline (see Recipes)
  33. Lysis buffer (see Recipes)

Equipment

Note: Biosafety level 2 (BSL 2) is required for culturing M. bovis BCG

  1. 37 °C incubator (humidified)/shaker (N-BIOTEK, model: NB-203QR /Eppendorf, New BrunswickTM Innova®, model: 40 )
  2. Spectrophotometer (Eppendorf, BioPhotometer®, model: D30 )
  3. 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)
  4. Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17R )
  5. Vortexer (Bio-rad, model: BR-2000 )
  6. Plate reader (reads in the visible spectrum, including 405 and 595 nm) (Tecan, Infinite®, model: m200 PRO )
  7. Bead beater (BioSpec Products, model: Mini-Beadbeater-16 )
  8. Heating block (heats to 95 °C) (NeoLab)
  9. Ultra low temperature freezer (Panasonic, model: MDF-U33V-PE )

Software

  1. GraphPad Prism 5

Procedure

  1. Culturing M. smegmatis
    1. Inoculate 50 μl of M. smegmatis mc2 155 strain from glycerol stock [32.5% glycerol (v/v) and 50 mM MgSO4 in 7H9-Glycerol-Tween 80 medium] in 5 ml Middlebrook 7H9 medium supplemented with 0.2% glycerol and 0.05% Tween 80 and grow at 37 °C with shaking at 200 rpm until the culture reaches saturation (OD600 ~4, usually takes 3-4 days). This culture serves as the primary inoculum.
    2. Inoculate 10 ml (for log phase, OD600 ~1) and 5 ml (for stationary phase, OD600 ~3) of Middlebrook 7H9 media supplemented with 0.2% glycerol and 0.05% Tween 80 with the primary inoculum such that the starting OD600 is 0.05 and grow at 37 °C with shaking at 200 rpm.
    3. Once the 10 ml culture reaches OD600 ~1 (usually takes ~14 h), pellet 9 ml of culture by centrifuging at 3,000 x g for 10 min in a 15 ml centrifuge tube at 4 °C. Collect 1 ml of culture supernatant in a 1.5 ml microcentrifuge tube and keep in ice. Keep the cell pellet on ice after carefully and completely removing the rest of the supernatant.
    4. When the 5 ml culture reaches OD600 ~3 (usually takes ~18-20 h), pellet 3 ml of culture by centrifuging at 3,000 x g for 10 min in a 15 ml centrifuge tube at 4 °C. Collect 1 ml of culture supernatant in a 1.5 ml microcentrifuge tube and keep in ice. Keep the cell pellet on ice after carefully and completely removing the rest of the supernatant.

  2. Culturing M. bovis BCG
    1. Inoculate 50 μl of M. bovis BCG from glycerol stock [32.5% glycerol (v/v) and 50 mM MgSO4 in 7H9-Glycerol-Tween 80-OADC medium] in 5 ml Middlebrook 7H9 medium supplemented with 0.2% glycerol and 0.05% Tween 80 containing 10% (v/v) OADC in a T25 tissue culture flask and grow at 37 °C in a humidified incubator as static cultures. Once the culture reaches saturation (OD600 ~4, usually takes 12-14 days), use it as the primary inoculum.
    2. Inoculate 20 ml (for log phase, OD600 ~1) and 10 ml (for stationary phase, OD600 ~3) of Middlebrook 7H9 media supplemented with 0.2% glycerol and 0.05% Tween 80 containing 10% (v/v) OADC with the primary inoculum such that the starting OD600 is 0.1 in T75 and T25 tissue culture flasks, respectively. Grow at 37 °C in a humidified incubator as static cultures.
    3. Once the 20 ml culture reaches OD600 ~1 (usually takes ~7-8 days), pellet 18 ml of culture by centrifuging at 3,000 x g for 10 min in a 50 ml centrifuge tube at 4 °C. Collect 1 ml of culture supernatant in a 1.5 ml microcentrifuge tube and keep in ice. Keep the cell pellet on ice after carefully and completely removing the rest of the supernatant.
    4. When the 10 ml culture reaches OD600 ~3 (usually takes ~14-15 days), pellet 6 ml of culture by centrifuging at 3,000 x g for 10 min in a 15 ml centrifuge tube at 4 °C. Collect 1 ml of culture supernatant in a 1.5 ml microcentrifuge tube and keep in ice. Keep the cell pellet on ice after carefully and completely removing the rest of the supernatant.

  3. Separating free and bound cAMP in the cytosolic fraction (identical for both M. smegmatis and M. bovis BCG) 
    1. Add 400 μl of cold phosphate buffered saline to the cell pellets and resuspend the cells either by vortexing or pipetting up and down several times. Transfer the cell suspension to a new 1.5 ml microcentrifuge tube.
    2. Pellet the cells by centrifuging at 3,000 x g for 10 min at 4 °C and discard the supernatant.
    3. Repeat the wash step once more as in steps C1 and C2
    4. Resuspend the cells in 400 μl lysis buffer.
    5. Add ~0.3 g glass beads to new bead beating vials and transfer the cell suspensions to the vials, respectively. Keep bead beating vials in ice.
    6. Lyse cells by bead beating (30 sec pulse with 3,450 oscillations/min, 6 times). Keep vials in ice between each cycle of bead beating.
    7. Centrifuge bead beating vials at 1,000 x g for 1 min at 4 °C and transfer the lysate to a new 1.5 ml microcentrifuge tube.
    8. Centrifuge the lysate at 17,000 x g for 20 min at 4 °C. Transfer the supernatant to a new 1.5 ml microcentrifuge tube and keep in ice. This serves as the cytosolic fraction.
    9. Protein concentration in the cytosolic fraction can be measured using a microplate-based Bradford method (Any other method can also be used.). Bovine serum albumin (BSA) is used as a standard in the Bradford assay.
    10. Transfer cytosolic fraction corresponding to 400 μg protein to an Amicon 0.5 ml 3 kDa cut-off centrifugal filter and centrifuge at 14,000 x g for 10-15 min at 4 °C. Collect 40 μl of the filtrate and transfer to a new 1.5 ml microcentrifuge tube and add equal volume of 0.2 N HCl (final concentration 0.1 N). Heat the sample for 10 min at 95 °C in a heating block and store at -70 °C until cAMP measurement. This fraction will provide the ‘free’ cAMP concentration in the cytosolic fraction.
      Note: The sample can be stored at -70 °C after heating at 95 °C if cAMP measurement is to be performed at a later point of time. Otherwise, the sample can be directly used to measure cAMP post heating.
    11. Collect 40 μl of the neat cytosolic fraction (from step C8) in a new 1.5 ml microcentrifuge tube and add equal volume of 0.2 N HCl (final concentration 0.1 N). Heat the sample for 10 min at 95 °C in a heating block and store at -70 °C until cAMP measurement. This fraction will provide the ‘total’ cAMP concentration in the cytosolic fraction.

  4. Separating free and bound cAMP in the culture supernatant (identical for both M. smegmatis and M. bovis BCG) 
    1. Transfer 400 μl of culture supernatants previously collected (see steps A3 and A4, B4 and B5 in Procedure) to Amicon 0.5 ml 3 kDa cut-off centrifugal filters and centrifuge at 14,000 x g for 10-15 min at 4 °C. Collect 150 μl of the filtrate and transfer to a new 1.5 ml microcentrifuge tube and add equal volume of 0.2 N HCl (final concentration 0.1 N). Heat the sample for 10 min at 95 °C in a heating block and store at -70 °C until cAMP measurement. This fraction will provide the ‘free’ extracellular cAMP concentration in the culture supernatant.
    2. Transfer 150 μl of culture supernatant (from steps A3 and A4, B4 and B5) to a new 1.5 ml microcentrifuge tube and add equal volume of 0.2 N HCl (final concentration 0.1 N). Heat the sample for 10 min at 95 °C in a heating block and store at -70 °C until cAMP measurement. This fraction will provide the ‘total’ extracellular cAMP concentration in the culture supernatant.

  5. Measurement of cAMP
    1. Take suitable aliquots of the samples and measure cAMP concentrations in each fraction either by radioimmunoassay (Dass et al., 2008) or ELISA procedures [kits can be used from Enzo Lifesciences (ADI-900-067), Abcam (ab65355) or Thermo Fisher Scientific (EMSCAMPL)].
    2. Express cytosolic ‘total’ (fraction collected in stepC11) and ‘free’ (fraction collected in stepC10) cAMP as ‘pmoles cAMP/100 μg protein’, and calculate the cytosolic ‘bound’ cAMP by subtracting the cAMP concentration in the ‘free’ from the concentration in the ‘total’.
    3. Express the extracellular ‘total’ (fraction collected in stepD2) and ‘free’ (fraction collected in stepD1) cAMP as ‘pmoles cAMP ml-1’, and calculate the extracellular ‘bound’ cAMP by subtracting the cAMP concentration in the ‘free’ from the concentration in the ‘total’.

Representative data


Figure 1. Free and bound cAMP in M. smegmatis. Free (white) and bound (gray) cAMP levels were measured from log phase cultures of M. smegmatis for both intracellular and extracellular fractions. The sum of free and bound levels represent the total cAMP. Mean and Standard Error of the Mean (SEM) were plotted for three biological replicates using GraphPad Prism 5. SEM was calculated by dividing the standard deviation (SD) by the square root of N, where N is the number of independent determinations. SD = , where  is the sample mean.

Notes

  1. Once cells are lysed, all steps should be carried out at 4 °C and samples kept in ice between all steps.
  2. As cAMP may dissociate from protein over time, the steps after lysis till the filtration with the Amicon membrane should be performed without delay.

Recipes

  1. Phosphate buffered saline
    0.8% NaCl
    0.02% KCl
    0.142% Na2HPO4
    0.024% KH2PO4
  2. Lysis buffer
    50 mM Tris-Cl (pH 8.2)
    100 mM NaCl
    10 mM 2-mercaptoethanol (2-ME)
    10% (v/v) glycerol
    1 mM phenylmethylsulphonyl fluoride (PMSF)

Acknowledgments

This protocol is adapted from the original method described in Banerjee et al. (2015). A.B. is supported by Senior Research Fellowship from the Council of Scientific & Industrial Research, Government of India. S.S.V. is a recipient of a J.C. Bose National Fellowship from the Department of Science & Technology, Government of India. This work was supported by the Department of Biotechnology, Government of India.

References

  1. 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.
  2. Dass, B. K., Sharma, R., Shenoy, A. R., Mattoo, R. and Visweswariah, S. S. (2008). Cyclic AMP in mycobacteria: characterization and functional role of the Rv1647 ortholog in Mycobacterium smegmatis. J Bacteriol 190(11): 3824-3834.
  3. Shenoy, A. R. and Visweswariah, S. S. (2006). New messages from old messengers: cAMP and mycobacteria. Trends Microbiol 14(12): 543-550. 

简介

分枝杆菌基因组编码涉及cAMP的合成,利用和降解的大量基因。例如,结核分枝杆菌H37Rv的基因组编码16个腺苷酸环化酶和10个携带环核苷酸结合(CNB)结构域的基因(Shenoy和Visweswariah,2006)。循环AMP由分枝杆菌有效分泌,细胞溶质以及细胞外cAMP水平可达数百微摩尔。我们最近报道,大量表达的普遍应激蛋白(USP; Rv1636在结核分枝杆菌H37Rv和MSMEG_3811分别在耻垢分枝杆菌中)分别结合cAMP(Banerjee等,2015)。鉴于存在于分枝杆菌中的cAMP结合蛋白的数量,预期细胞内cAMP的显着部分可能与蛋白质结合。通常用于测量cAMP的方法是放射免疫测定(RIA)和ELISA。然而,这些方法包括将cAMP“结合”解离成蛋白质的样品的预先酸化,因此代表样品中存在的“总”cAMP。在本协议中,我们描述了一种将cAMP'结合'蛋白质与蛋白质“自由”分离或与蛋白质不相关的方法。这通过使细胞溶质级分或培养物上清液通过具有3kDa截止值的膜过滤来进行。只有'自由'cAMP才能通过膜。因此,滤液中的cAMP浓度代表样品中的“游离”cAMP。原始细胞溶质级分或培养上清液中的环AMP水平代表“总”cAMP浓度。从“总”中减去“自由”提供了与蛋白质结合的cAMP量。

关键字:营, cAMP结合蛋白, 结核分枝杆菌, 普遍的应激蛋白

材料和试剂

  1. 15ml聚丙烯离心管(Thermo Fisher Scientific,目录号:339650),
  2. 1.5ml微量离心管(Corning,Axygen ,目录号:MCT-175-C)
  3. T25聚苯乙烯组织培养处理的烧瓶(Nest Biotechnology,目录号:707003)
  4. T75聚苯乙烯组织培养处理的烧瓶(Nest Biotechnology,目录号:708003)
  5. 带有Ultracel-3膜的Amicon Ultra-0.5ml 3-kDa离心过滤器(Merck Millipore Corporation,目录号:UFC500324)
  6. 50ml聚丙烯离心管(Thermo Fisher Scientific,目录号:339652),
  7. 用于生长5和10 ml培养物的玻璃试管
  8. 96孔聚苯乙烯板(Sigma-Aldrich,产品号:M2936)
  9. 0.5mm直径的玻璃珠(Bio Spec Products Inc,目录号:11079105)
  10. 2ml聚丙烯珠粒小瓶(Bio Spec Products Inc,目录号:10831)
  11. 耻垢分枝杆菌 mc 2 155(ATCC,目录号:700084)
  12. < em>牛分枝杆菌BCG(ATCC,目录号:35734)
  13. Middlebrook 7H9培养基(BD Biosciences,目录号:271310)
  14. 甘油(Thermo Fisher Scientific,目录号:17904)
  15. 硫酸镁(Merck Millipore Corporation,目录号:105886)
  16. 吐温80(Sigma-Aldrich,产品编号:P4780)
  17. 7H9-甘油 - 吐温80培养基
  18. 油酸/白蛋白/葡萄糖/过氧化氢酶(OADC)生长补充剂(BD Biosciences,目录号:211886)
  19. Bradford试剂(Sigma-Aldrich,目录号:B6916)
  20. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
  21. 盐酸(HCl)(Fisher Scientific,目录号:A144SI-212)
  22. cAMP ELISA试剂盒(Enzo Lifesciences,目录号:ADI-900-067)
  23. cAMP直接免疫测定试剂盒(Abcam plc,目录号:ab65355)
  24. 循环AMP竞争性ELISA试剂盒(Thermo Fisher Scientific,目录号:EMSCAMPL)
  25. 氯化钠(Merck Millipore Corporation,目录号:1064040500)
  26. 氯化钾(Merck Millipore Corporation,目录号:1049360500)
  27. 磷酸氢二钠(Merck Millipore Corporation,目录号:1065860500)
  28. 磷酸二氢钾(Merck Millipore Corporation,目录号:1048730250)
  29. 三(羟甲基)氨基甲烷(Merck Millipore Corporation,目录号:1083820500)
  30. 2-巯基乙醇(Sigma-Aldrich,产品号:M6250)
  31. 苯甲基磺酰氟(Sigma-Aldrich,产品编号:PMSF-RO ROCHE)
  32. 磷酸盐缓冲盐水(见配方)
  33. 裂解缓冲液(见配方)

设备

注意:培养牛分枝杆菌BCG 需要生物安全2级(BSL 2)

  1. 37℃培养箱(加湿的)/振荡器(N-BIOTEK,型号:NB-203QR/Eppendorf,New Brunswick ,型号:40)
  2. 分光光度计(Eppendorf,BioPhotometer ,型号:D30)
  3. 装备有TX-400摇摆转子(Thermo Fisher Scientific,型号:TX-400)的冷冻离心机(Thermo Fisher Scientific,Heraeus?sup?Megafuge TM型号:16R)适配器(数字20& 23)
  4. 微量离心机(Thermo Fisher Scientific,型号:MicroCL 17R)
  5. Vortexer(Bio-rad,型号:BR-2000)
  6. 读板器(读入可见光谱,包括405和595nm)(Tecan,Infinite ,型号:m200 PRO)
  7. Bead beater(BioSpec Products,型号:Mini-Beadbeater-16)
  8. 加热块(加热至95°C)(NeoLab)
  9. 超低温冷冻箱(Panasonic,型号:MDF-U33V-PE)

软件

  1. GraphPad Prism 5

程序

  1. 培养M.耻垢
    1. 接种50微升的M.来自甘油储备液[在7H9-甘油-Tween 80培养基中的32.5%甘油(v/v)和50mM MgSO 4]中的耻垢分枝杆菌mc 2 155菌株] 5ml补充有0.2%甘油和0.05%吐温80的Middlebrook 7H9培养基,并在37℃下以200rpm振荡生长直到培养物达到饱和(OD 600?4,通常需要3-4天)。这种培养物作为主要的接种物
    2. 将补充有0.2%甘油的Middlebrook 7H9培养基接种10ml(对数期,OD 600±1)和5ml(对于稳定期,OD 600?3) 0.05%Tween 80与初级接种物混合,使得起始OD 600为0.05,并在37℃下以200rpm振摇生长。
    3. 一旦10ml培养物达到OD 600?1(通常需要?14小时),通过在15ml离心机中以3,000xg离心10分钟来沉淀9ml培养物管在4℃。收集1毫升培养上清液在1.5毫升微量离心管,并保存在冰中。小心地将细胞沉淀放在冰上,并彻底清除剩余的上清液
    4. 当5ml培养物达到OD 600-3(通常需要?18-20小时)时,通过在15℃以3,000xg离心10分钟沉淀3ml培养物ml离心管在4℃。收集1毫升培养上清液在1.5毫升微量离心管,并保存在冰中。小心地将细胞沉淀放在冰上,并彻底清除剩余的上清液
  2. 培养M. bovis BCG
    1. 接种50微升的M.来自甘油储液[在7H9-甘油-Tween 80-OADC培养基中的32.5%甘油(v/v)和50mM MgSO 4]的牛血清/牛血清BCG)的5ml Middlebrook 7H9培养基%甘油和含有10%(v/v)OADC的0.05%吐温80中,并在37℃下在加湿培养箱中作为静态培养物生长。一旦培养物达到饱和(OD <600> 4,通常需要12-14天),将其用作主要接种物。
    2. 接种补充有0.2%甘油的Middlebrook 7H9培养基(对数期,OD 600±1)和10ml(对于稳定期,OD 600?3) 0.05%吐温80(含有10%(v/v)OADC)与初级接种物混合,使得在T75和T25组织培养烧瓶中的起始OD 600为0.1。在37℃下在湿润的培养箱中作为静态培养物生长
    3. 一旦20ml培养物达到OD 600-1(通常需要?7-8天),通过在50℃下以3,000xg离心10分钟沉淀18ml培养物ml离心管在4℃。收集1毫升培养上清液在1.5毫升微量离心管,并保存在冰中。小心地将细胞沉淀放在冰上,并彻底清除剩余的上清液
    4. 当10ml培养物达到OD 600?3(通常需要?14-15天)时,通过在15℃以3,000xg离心10分钟来沉淀6ml培养物ml离心管在4℃。收集1毫升培养上清液在1.5毫升微量离心管,并保存在冰中。小心地将细胞沉淀放在冰上,并彻底清除剩余的上清液
  3. 在胞质部分中分离游离和结合的cAMP(对于耻垢分枝和 牛 BCG都是相同的)
    1. 加入400微升冷磷酸盐缓冲盐水的细胞沉淀和重悬细胞通过涡旋或吸移上下数次。将细胞悬液转移到新的1.5 ml微量离心管中
    2. 通过在4℃下以3000xg离心10分钟来沉淀细胞,并丢弃上清液。
    3. 重复洗涤步骤一次,如步骤C1和C2
    4. 将细胞重悬在400μl裂解缓冲液中
    5. 将约0.3g玻璃珠添加到新的珠打孔小瓶中,并将细胞悬浮液分别转移到小瓶中。将珠子小瓶保持在冰上。
    6. 裂解细胞通过珠打(30秒脉冲与3,450振荡/分钟,6次)。在每次珠磨循环之间保持瓶子在冰上。
    7. 在4℃下将珠子在1000×g下离心1分钟,并将裂解物转移到新的1.5ml微量离心管中。
    8. 在4℃下以17,000×g离心裂解物20分钟。转移上清液到一个新的1.5毫升微量离心管,并保持在冰。这作为胞质部分。
    9. 可以使用基于微板的Bradford方法(也可以使用任何其他方法)测量胞质部分中的蛋白质浓度。牛血清白蛋白(BSA)用作Bradford测定中的标准品
    10. 将对应于400μg蛋白的细胞溶质级分转移到Amicon 0.5ml 3kDa截止离心过滤器,并在4℃下以14,000×g离心10-15分钟。收集40μl滤液,转移到新的1.5ml微量离心管中,加入等体积的0.2N HCl(终浓度0.1N)。在加热块中在95℃下加热样品10分钟,并储存在-70℃下直到cAMP测量。该部分将提供胞质部分中的"游离"cAMP浓度 注意:如果在稍后的时间点进行cAMP测量,则样品可以在95℃加热后储存在-70℃。否则,样品可以直接用于测量cAMP后加热。
    11. 在新的1.5ml微量离心管中收集40μl纯胞质部分(来自步骤C8)并加入等体积的0.2N HCl(终浓度0.1N)。在加热块中在95℃下加热样品10分钟,并储存在-70℃下直到cAMP测量。该部分将提供胞质部分中的'总'cAMP浓度
  4. 在培养上清液中分离游离和结合的cAMP(对于耻垢分枝杆菌和牛分枝杆菌 BCG是相同的)
    1. 将400μl先前收集的培养物上清液(参见步骤中的步骤A3和A4,B4和B5)转移到Amicon 0.5ml 3kDa截止离心过滤器中,并在14,000×g离心10-15分钟4℃。收集150μl滤液,转移到新的1.5ml微量离心管中,加入等体积的0.2N HCl(终浓度0.1N)。在加热块中在95℃下加热样品10分钟,并储存在-70℃下直到cAMP测量。该级分将在培养上清液中提供"游离"细胞外cAMP浓度
    2. 转移150微升培养上清液(从步骤A3和A4,B4和B5)到一个新的1.5毫升微量离心管,加入等体积的0.2 N HCl(终浓度0.1 N)。在加热块中在95℃下加热样品10分钟,并储存在-70℃下直到cAMP测量。该部分将提供培养上清液中的"总"细胞外cAMP浓度
  5. cAMP测量
    1. 取适当的样品等分试样,并通过放射免疫测定(Dass等人,2008)或ELISA程序测量各级分中的cAMP浓度[试剂盒可以使用Enzo Lifesciences(ADI-900-067), Abcam(ab65355)或Thermo Fisher Scientific(EMSCAMPL)]
    2. 将'游离'(在步骤C11中收集的级分)和'游离'(在步骤C10中收集的级分)cAMP作为'pmoles cAMP /100μg蛋白',计算细胞溶质'总'从"总"的浓度
    3. 将在步骤D2中收集的细胞外"总"(在步骤D2中收集的部分)和"游离"(在步骤D1中收集的部分)cAMP表示为"pmoles cAMP ml sup-1",并通过减去cAMP浓度在'游离'中的浓度与'总'的浓度

代表数据


图1. M中的自由和结合cAMP。耻垢 。从M的对数期培养物测量游离(白色)和结合(灰色)cAMP水平。耻垢分泌物对于细胞内和细胞外部分。游离和结合水平的总和代表总cAMP。使用GraphPad Prism 5对三个生物重复绘制平均值的平均值和标准误差(SEM)。通过用N的平方根除以标准偏差(SD)计算SEM,其中N是独立测定的数目。 SD = ,其中  是样本均值。

笔记

  1. 一旦细胞裂解,所有步骤应在4°C进行,样品在所有步骤之间保持冰
  2. 由于cAMP可随时间从蛋白质上解离,裂解后直到用Amicon膜过滤的步骤应该毫无延迟地进行。

食谱

  1. 磷酸盐缓冲盐水
    0.8%NaCl
    0.02%KCl
    0.142%Na 2 HPO 4
    0.024%KH 2 PO 4 sub/
  2. 裂解缓冲液
    50mM Tris-Cl(pH8.2) 100 mM NaCl
    10mM 2-巯基乙醇(2-ME) 10%(v/v)甘油 1mM苯甲基磺酰氟(PMSF)

致谢

  1. 该协议改编自Banerjee等人中描述的原始方法。 (Banerjee等人,2015)。 A.B.得到科学技术委员会高级研究奖学金的支持。印度政府工业研究。 S.S.V.是科学与工程学院J.C.Bose国家奖学金的接受者。技术,印度政府。这项工作得到印度政府生物技术司的支持。

参考文献

  1. Banerjee,A.,Adolph,RS,Gopalakrishnapai,J.,Kleinboelting,S.,Emmerich,C.,Steegborn,C.and Visweswariah,SS(2015)。  分枝杆菌中的通用胁迫蛋白(USP)结合cAMP。 J Biol Chem 290(20):12731-12743。
  2. Dass,BK,Sharma,R.,Shenoy,AR,Mattoo,R.and Visweswariah,SS(2008)。  分枝杆菌中的环AMP:Rv1647直向同源物在耻垢分枝杆菌中的表征和功能作用 190(11):3824-3834。
  3. Shenoy,AR and Visweswariah,SS(2006)。  新来自旧信使的消息:cAMP和分枝杆菌。趋势微生物 14(12):543-550。

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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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.
  2. 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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