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本实验方案简略版
Apr 2019

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An in vitro Assay to Screen for Substrates of PKA Using Phospho-motif-specific Antibodies
利用磷酸基团特异性抗体体外筛选PKA底物   

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Abstract

Kinases function as regulators of many cellular processes such as cell migration. These enzymes typically phosphorylate target motif sequences. Mass spec or phospho-specific antibody detection can be used to determine whether a kinase can phosphorylate proteins of interest, however, mass spec can be expensive and phospho-antibodies for the protein of interest may not exist. In this protocol, we will describe an in vitro kinase assay to provide a preliminary readout on whether a protein of interest may be phosphorylated by PKA. Our protein of interest is purified after expression in bacteria and treated with recombinant PKA from bovine heart. Protein is then extracted and a western blot is performed using a phospho-specific antibody for PKA’s target motif. This will allow us to quickly determine if it is possible for PKA to phosphorylate our protein of interest.

Keywords: Protein Kinase A (PKA) (蛋白激酶A), Kinase activity (激酶活性), In vitro (体外), Western blot (免疫印迹), Protein phosphorylation (蛋白磷酸化)

Background

Hormones and other factors that cause activation of adenylate cyclase through G-linked G-protein coupled receptors (GPCR) and subsequently promote generation of second messenger cAMP can affect cellular processes like cell migration. Elevation of cAMP level leads to activation of PKA, a serine-threonine kinase that plays an important role in the regulation of actin cytoskeletal dynamics in migrating cells. PKA influences different facets of actin cytoskeleton-regulatory processes including modulation activities of a) Rho-family GTPases (Rho, Rac and Cdc42), b) actin-binding proteins (e.g., VASP [vasodilator stimulated phosphoprotein]), c) kinases which indirectly control the function of actin-binding proteins (e.g., p21-activated kinase) and d) myosin (Howe, 2004). PKA and other kinases, however, can also modulate other proteins by phosphorylation that are currently unknown.

There are multiple ways to determine kinase activity on a protein of interest such as mass spec for phosphopeptides, however, such assays can be costly. In this protocol, we will describe the use of an in vitro kinase assay to examine PKA kinase activity on a GST-tagged protein of interest using a phospho-specific antibody for PKA’s target motif. Using a PKA motif antibody is advantageous if known phosphorylation sites are unknown on a protein or if no phosphospecific protein antibody exists. While this assay is quick and simple to perform, it depends on the kinase targeting a motif pre-determined by available antibodies and thus limited in potential. A different kinase can be easily tested if an antibody to detect that kinase’s phospho motif exists. Despite this, successful readout from this assay can provide motivation to pursue more comprehensive exploration into kinase phosphorylation of a protein of interest. Although this protocol describes usage of recombinant protein purified from bacteria, protein can be obtained from any source (i.e., insect or human cells) but total yield of protein will vary dependent on the source and may need to be optimized. Figure 1 below illustrate the general workflow for this protocol.



Figure 1. General protocol workflow for kinase analysis

Materials and Reagents

  1. Eppendorf tubes
  2. Bacteria culture tube (Southern Labware, catalog number: 110178 )
  3. BL21 competent E. coli (New England BioLabs, catalog number: C2530H )
  4. LB Broth (Thermo Fisher, catalog number: 10855001 )
  5. LB Agar Ampicillin-100 (Sigma-Aldrich, catalog number: L5667 )
  6. IPTG (Sigma-Aldrich, catalog number: I6758 )
  7. Ampicillin 100 mg/ml (Sigma-Aldrich, catalog number: A5354 )
  8. Glutathione Agarose (Thermo Fisher, Pierce, catalog number: 16100 )
  9. Protease inhibitor cocktail for bacteria (Sigma-Aldrich, catalog number: P8465 )
  10. pGEX-4T1 GST-fusion vector (Sigma-Aldrich, catalog number: GE28-9545-49 )
  11. PBS (Lonza, BioWhittaker, catalog number: BW17516F )
  12. IP Lysis Buffer (Thermo Fisher, Pierce, catalog number: 87787 )
  13. PKA (Sigma-Aldrich, catalog number: P2645-400UN )
  14. Laemmli SDS sample buffer, reducing 6x (Alfa Aesar, catalog number: J61337-AC )
  15. BES (Thermo Fisher, BioReagents, catalog number: BP501500 )
  16. EGTA (Tocris Bioscience, catalog number: 28-071-G )
  17. MgCl2 (Thermo Fisher, catalog number: AB0359 )
  18. ATP (Sigma-Aldrich, catalog number: A7699 )
  19. Phosphocreatine (Sigma-Aldrich, catalog number: P7936 )
  20. DTT (Sigma-Aldrich, catalog number: D9779 )
  21. Prestained protein ladder (Thermo Fisher, catalog number: 26616 )
  22. 15% Criterion Tris-HCl protein gel (Bio-Rad, catalog number: 3450019 )
  23. 10x Tris/Tricine/SDS running buffer (Bio-Rad, catalog number: 1610744 )
  24. 10x Tris/Glycine transfer buffer (Bio-Rad, catalog number: 1610734 )
  25. Methanol (Thermo Fisher, Fisher Chemical, catalog number: A412-500 )
  26. Nitrocellulose membrane (Bio-Rad, catalog number: 1620115 )
  27. TBS-T (Bio-Rad, catalog number: BUF028 )
  28. BSA (Thermo Fisher, BioReagents, catalog number: BP1600100 )
  29. Phospho-PKA subtract RRXS antibody (Cell Signaling Technology, catalog number: 9624 )
  30. Peroxidase Goat Anti-Rabbit IgG (Jackson ImmunoResarch, catalog number: 111-035-144 )
  31. Clarity ECL (Bio-Rad, catalog number: 1705060 )
  32. Kinase buffer (see Recipes)
  33. IPTG solution (see Recipes)

Equipment

  1. Refrigerated Centrifuge for Eppendorf tubes (Sorvall, Legend RT, SO-LEGRT)
  2. Eppendorf tube heater (Lab Line, Multi Blok Heater, LV40429530)
  3. Shaking incubator set to 37 °C (Thermo Scientific, MaxQ 5000, SHKE50007)
  4. Waterbath or drybath set to 37 °C (Boekel Scientific, Small Water Bath, 290100)
  5. Sonicator (Bransonic, Ultrasonic Baths, CPX-952-116R)
  6. Rotator for Eppendorf tube (Boekel Scientific, Scientific Rotating Tube Rotator, UX-51202-00)
  7. Microcentrifuge (Thermo Scientific, accuSpin Micro 17, 13-100-676)
  8. Mini Trans-Blot (Bio-Rad)
  9. Mini-PROTEAN Tetra cell (Bio-Rad)
  10. ChemiDoc XRS+ System (Bio-Rad)

Software

  1. ImageLab (Bio-Rad)

Procedure

  1. Transformation
    1. Thaw BL21 competent cells on ice.
    2. Combine 50 μl of BL21 cells with 100 ng of plasmid that can be expressed/induced in bacteria (i.e., contains laclq gene sequence which prevents expression until induced by IPTG), in our case, GST vector (pGEX-4T1) with gene of interest (in our case, Profilin-1) and incubate on ice for 30 min.
    3. Heat shock mixture at 42 °C for 45 s using water bath or heat block.
    4. Place mixture back on ice for 2 min.
    5. Incubate bacteria mixture in 200 μl of LB broth in shaking incubator for 1 h at 300 rpm and 37 °C (make sure mixture is not in an airtight tube).
    6. Plate all the mixture onto LB agar plate containing appropriate antibiotics, in our case, ampicillin (final concentration 100 μg/ml) and spread until plate is dry.
    7. Incubate at 37 °C overnight (ensure plate is inverted).

  2. Protein induction and extraction
    1. Pick a single colony from bacteria plate and grow in 3 ml of LB broth and appropriate antibiotic overnight (ampicillin final concentration 100 μg/ml) in bacteria culture tube in shaking incubator at 37 °C (ensure tube is not airtight).
    2. Take 1 ml of this solution and grow in 44 ml of LB broth and appropriate antibiotic (ampicillin final concentration 100 μg/ml) at 37 °C in rotating incubator at 300 rpm for 3 h.
    3. Add 1:1,000 100 mM IPTG to induce expression to mixture and incubate at 300 rpm for another 3 h.
    4. Transfer bacteria solution into a 50 ml centrifuge tube and centrifuge bacteria culture for 20 min at 3,500 x g and 4 °C to harvest bacteria.
    5. Discard supernatant.
    6. Resuspend bacteria pellet in 850 μl of IP lysis buffer with 50 μl/ml of bacterial protease inhibitor and transfer into a microcentrifuge Eppendorf tube. Bacterial protease inhibitor should be prepared and used fresh each time.
    7. Sonicate for 10 s (no heat), rest on ice for 2 min, and repeat 2 more times. This step is used to shear DNA and break bacteria.
    8. Centrifuge at 17,000 x g for 30 min at 4 °C.
    9. Collect supernatant.
      Note: Product can be run on a gel and Coomassie stained to confirm protein expression.

  3. Protein purification and preparation for kinase treatment
    1. Aliquot ~400 μl of Glutathione Agarose (GST bead slurry) to Eppendorf tubes.
    2. Wash two times with 500 μl cold PBS and centrifuge beads down between washes at 8,000 x g for 1 min.
    3. Wash one time with 500 μl IP lysis buffer and centrifuge beads down at 8,000 x g for 1 min.
    4. Add IP lysis buffer to make 50/50 slurry of beads and IP lysis buffer.
    5. Transfer 160 μl of this slurry into a new Eppendorf tube and add 540 μl of IP lysis buffer to it and 50 μl/ml bacterial protease inhibitor. Bacterial protease inhibitor should be prepared and used fresh each time.
    6. Add 300 μl of bacteria lysate and rotate using rotating end-over-end rotator at 30 rpm for 2 h at 4 °C.
    7. Centrifuge beads down at 8,000 x g for 1 min and wash three times with 500 μl IP lysis buffer and centrifuge beads down between washes at 8,000 x g for 1 min.
    8. Wash two times with 500 μl kinase buffer and centrifuge beads down between washes at 8,000 x g for 1 min (leave at 50/50 slurry).

  4. Kinase treatment
    1. Reconstitute PKA by adding 200 μl of kinase buffer (400UN of PKA comes as a dry pellet) and let sit for 20 min at room temp, this is your PKA solution.
    2. Aliquot 2 Eppendorf tubes with 40 μl of GST bead solution containing protein of interest from previous section. One tube will be used as a negative control.
    3. Add 5 μl of PKA solution to one tube (aliquot the rest of the PKA solution into 5 μl and store at -80 °C for future use) and 5 μl of kinase solution (i.e., kinase solution with no PKA) to the other. Note that PKA is susceptible to freeze-thaw and loses some activity after the first freeze. The recommended aliquot amount is enough for one treatment group each time and thus no need to freeze-thaw. PKA should not be used after being frozen for > 1 year in -80 °C.
    4. Incubate at 32 °C for 1 h using water bath or heat block and mix using vortex every 15 min to ensure beads are not settled to bottom of tube.
    5. Wash two times with 500 μl IP lysis buffer buffer and centrifuge beads down between washes at 8,000 x g for 1 min and leave at 50/50 slurry.
    6. Aliquot 30 μl of solution to a new tube and add 60 μl of 1.5x Laemilli buffer.
    7. Boil for 5 min and centrifuge at 17,000 x g for 5 min.
    8. Transfer supernatant to a fresh microcentrifuge Eppendorf tube.

  5. Western blot
    1. Load 5 μl of protein ladder into 15% gel and load 30 μl of protein sample (supernatant from last step in previous section) into adjacent wells.
    2. Fill PROTEAN with 1x running buffer and run at 200 V for ~45 min (might be more or less time, run until blue dye from sample buffer runs off the bottom of the gel). These settings were used for our protein-of-interest, settings may be different for other proteins.
    3. Transfer protein from gel onto either PVDF or nitrocellulose membrane (we used nitrocellulose) by using Trans-Blot filled with 1x transfer buffer (80% transfer buffer, 20% methanol) at 100 V for ~1.5 h (might be more or less depending on size of protein of interest). Make sure gel and membrane are placed in the correct orientation, i.e., the gel should be closer to the negative charge source (typically the black colored part of the transfer cassette should be closest to the gel). These settings were used for our protein-of-interest, settings may be different for other proteins.
    4. Cut membrane around protein of interest (1-2 ladder bands above and below protein weight) and block with 5% BSA in TBS-T for 30 min at room temperature with rocking motion. We used an empty microscope coverslip slide box to incubate our membranes (total blocking buffer volume 1.5 ml). In our case, GST-Pfn1 has a molecular weight of ~41 kd, so membrane was cut between 30 and 70 kd. Depending on the mass of your protein-of-interest, a different section may need to be cut based off the protein ladder.
    5. Incubate overnight 5% BSA in TBS-T with 1:1,000 pRRXS substrate antibody at 4 °C with rocking motion at 10 rpm. Using the same vessel as described previously, we use 1.5 ml of blocking solution and 1.5 μl of antibody.
    6. Wash with TBS-T one time for 5 min.
    7. Incubate with 5% BSA in TBS-T for 1 h at room temp with 1:1,000 anti-rabbit peroxidase antibody with rocking motion at 10 rpm. Using the same vessel as described previously, we use 1.5 ml of blocking solution and 1.5 μl of antibody.
    8. Wash 3-4 times with TBS-T for 5 min each wash.
    9. Add Clarity ECL solution to membrane and incubate for 1 min at room temperature.
    10. Image membrane (example results shown in Figure 2, PKA treated GST-Pfn1 is detected by pRRXS antibody, using a BioRad ChemiDoc).


      Figure 2. pRRXS antibody detects PKA phosphorylated GST-Pfn1. GST-Pfn1 is phosphorylated by PKA and detected by pRRXS antibody whereas kinase buffer solution treated GST-Pfn1 (negative control) is not detected. A Coomassie from this sample is shown as well to illustrate the amount of pulldown to obtain the phospho-band.

Data analysis

  1. Compare lane from protein sample treated with PKA and sample not treated with PKA.
  2. If band intensity is higher in PKA treated sample, the sample has increased phosphorylation as per pRRXS phosphospecific PKA antibody (in our case, in Figure 2, we saw no band intensity to full band intensity after treatment with PKA).

Recipes

  1. Kinase buffer
    Final concentration of all components is listed below:
    20 mM BES pH 7 (starts as powder, dilute in water and pH correct until appropriate concentration). This is used as the base of the Kinase buffer
    20 mM EGTA (starts as powder, add into BES solution)
    6 mM MgCl2 (starts as powder, add into BES solution)
    5 mM ATP (starts as powder, add into BES solution)
    10 mM phosphocreatine (starts as powder, add into BES solution)
    1 mM DTT (starts as powder, add into BES solution)
  2. IPTG solution
    Make 100 mM IPTG solution using water and IPTG powder and store at -20 °C

Acknowledgments

This work was supported by a grant from the National Institute of Health (2R01CA108607) to PR. David Gau was supported by a National Science Foundation pre-doctoral fellowship (2012139050) and an NIH Cardiovascular Bioengineering pre-doctoral training grant (2T32HL076124 to DG).

Competing interests

The authors have no competing interests to report.

References

  1. Howe, A. K. (2004). Regulation of actin-based cell migration by cAMP/PKA. Biochim Biophys Acta 1692(2-3): 159-174.

简介

[摘要 ] 激酶可充当许多细胞过程(例如细胞迁移)的调节剂,这些酶通常可磷酸化靶基序序列,质谱或磷酸特异性抗体检测可用于确定激酶是否可磷酸化目标蛋白。能成为规格昂贵和磷酸化抗体蛋白的兴趣可能就不存在。在这个协议中,我们将介绍一种在体外 激酶测定法可提供有关目的蛋白是否可被PKA磷酸化的初步读数。我们的目的蛋白在细菌中表达后纯化,并用牛心脏的重组PKA处理,然后提取蛋白,并使用蛋白印迹进行蛋白质印迹磷酸化针对PKA靶标的特异性抗体,这将使我们能够快速确定PKA是否可能使我们感兴趣的蛋白质磷酸化。

[背景 ] 激素和其他因素会通过G连接的G蛋白偶联受体(GPCR)激活腺苷酸环化酶并随后促进第二信使cAMP的产生,从而影响细胞迁移等细胞过程。cAMP水平升高会导致PKA激活,丝氨酸-苏氨酸激酶在迁移细胞中肌动蛋白细胞骨架动力学的调节中起着重要作用.PKA影响肌动蛋白细胞骨架调节过程的不同方面,包括a)Rho-fa 微小GTPases (Rho,Rac 和Cdc42)的调节活性, B)肌动蛋白结合蛋白(例如,VASP [血管舒张兴奋剂的UI Ated磷蛋白]),C)激酶间接地控制肌动蛋白结合蛋白(的作用例如,P21活化激酶)和d)肌球蛋白(豪,2004) 。但是,PKA和其他激酶也可以通过磷酸化调节其他蛋白质,目前尚不知道。

有M 的UI Tiple方法来确定激酶活性蛋白质上的一些景点如质谱对磷酸肽,然而,这样的分析可能是昂贵的。在这个协议中,我们将介绍使用的体外激酶试验来检查PKA激酶交流使用磷酸特异性抗体对PKA的目标基序进行感兴趣的GST标记蛋白的纯化。如果蛋白上已知的磷酸化位点未知或不存在磷酸特异性蛋白抗体,则使用PKA基序抗体是有利的。简单的执行,这取决于激酶靶向母题预先确定受可用抗体,从而限制了潜在可能不同激酶可以很容易地测试如果抗体来检测激酶'S 磷基序的存在。尽管这样,Successf ü 大号读数从该测定法可以提供动机,以便对目标蛋白的激酶磷酸化进行更全面的探索。然而,该方案描述了重组蛋白纯化的用途 细菌指明分数,蛋白质可以得到来自任何来源(即,昆虫和人类细胞),但总产量蛋白质会有所不同取决于源,可能需要进行优化,图1,下面说明一般的工作流程,此协议。



图茜1.一般Protoc 011工作流程激酶分析

关键字:蛋白激酶A, 激酶活性, 体外, 免疫印迹, 蛋白磷酸化

材料和试剂


 


1. Eppendorf管      


2. 细菌培养桶e(Southern Labware,目录号:110178)      


3. BL21主管大肠杆菌(新英格兰生物实验室,目录编号:C2530H)      


4. LB汤(Thermo Fisher,商品目录号:108500001)      


5. LB琼脂氨苄青霉素100(Sigma-Aldrich,目录号:L5667)      


6. IPTG(Sigma-Aldrich,目录号:I6758)      


7. 氨苄西林100 mg / ml(西格玛奥德里奇,目录号:A5354)      


8. 谷胱甘肽琼脂糖(Thermo Fisher,Pierce,目录号:16100)      


9. 细菌的蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8465)      


10. PGEX-4T1 GST-融合载体(Sigma-Aldrich公司,目录号:GE28-9545-49)   


11. PBS(龙沙,Bio Whittaker公司,目录号:BW17516F)   


12. IP裂解缓冲液(Thermo Fisher,Pierce,目录号:87787)   


13. PKA(Sigma-Aldrich,目录号:P2645-400UN)   


14. Laemmli SDS样本缓冲区,减少了6倍(Alfa Aesar ,目录号:J61337-AC)   


15. BES(Thermo Fisher,BioReagents ,目录号:BP501500)   


16. EGTA(Tocris Bioscience,目录号:28-071-G)   


17. MgCl 2 (Thermo Fisher,目录号:AB0359)   


18. ATP(西格玛奥德里奇,目录号:A7699)   


19. 磷酸肌酸(Sigma-Aldrich,目录号:P7936)   


20. DTT(西格玛奥德里奇,目录号:D9779)   


21. 预制蛋白梯(Thermo Fisher,目录号:26616)   


22. 15%标准的Tris-HC 升蛋白凝胶(Bio-Rad公司,目录编号:3450019)   


23. 10x Tris / Tricine / SDS运行缓冲液(Bio-Rad,目录号:1610744)   


24. 10X的Tris /甘氨酸转移缓冲液(Bio-Rad公司,目录编号:1610734)   


25. 甲醇(Thermo Fisher,Fisher Chemical,目录号:A412-500)   


26. 硝酸纤维素膜(Bio-Rad,目录号:1620115)   


27. TBS-T(伯乐,目录号:BUF028)   


28. BSA(赛默飞世尔,生物试剂,目录号:BP1600100)   


29. 磷酸-PKA减去RRXS抗体(Cell Signaling Technology,目录号:9624)   


30. 过氧化物酶山羊抗兔IgG(Jackson ImmunoResarch ,目录号:111-035-144)   


31. Clarity ECL(Bio-Rad,目录号:1705060)   


32. 激酶缓冲液(请参阅食谱)   


33. IPTG解决方案(请参阅食谱)   


配套设备


 


Eppendorf管冷冻离心机(Sorvall ,Legend RT,SO-LEGRT)
Eppendorf管式加热器(实验室管线,多段加热器,LV40429530)
摇动培养箱设置为37°C(Thermo Scientific,MaxQ 5000,SHKE50007)
水浴或干浴温度设置为37°C(Boekel Scientific,小型水浴,290100)
超声波仪(布兰尼克超声浴,CPX-952-116R)
Eppendorf管旋转器(Boekel Scientific,科学旋转管旋转器,UX-51202-00)
微量离心机(Thermo Scientific,accuSpin Micro 17,13-100-676)
迷你转运蛋白(Bio-Rad)
Mini-PROTEAN Tetra细胞(Bio-Rad)
ChemiDoc XRS +系统(Bio-Rad)
 


软体类


 


ImageLab(Bio-Rad)
 


程序


 


转型
在冰上解冻BL21感受态细胞。
结合50 Myueru BL21细胞与100毫微克质粒可以表达/诱导细菌(即,包含Laclq 基因序列中防止表达直到IPTG诱导),在本例中,GST载体(pGEX-4T1)随着基因的兴趣(本例中为Profilin-1),并在冰上孵育30分钟。
使用水浴或加热块在42 °C加热冲击混合物45 s。
将混合物放回冰上2分钟。
将细菌混合物孵育200 MYU大号中LB肉汤在振荡培养箱1个小时以300rpm和37℃(确保混合物不是在气密管)。
该混合物所有镀到LB琼脂平板含适当的抗生素,在我们这里,氨苄青霉素(终浓度为100 MYU 摹/ ml)和传播直到板干。
在37°C下孵育过夜(确保将板倒置)。
 


蛋白质诱导和提取
单菌落从这里选择细菌板并生长在第3 毫升中LB肉汤和合适的抗生素Overnig 高程(氨苄青霉素终浓度100 MYU ģ / ml)的细菌培养管在振荡培养箱中于37℃(确保管不是气密的)。
1送ML 中此溶液中并生长在44毫升LB肉汤和合适的抗生素(氨苄青霉素终浓度100 MYU ģ / ml)的在37℃下在旋转恒温箱中于300rpm下3 H.
加入1:1,000 100 mM IPTG以诱导表达到混合物中,并以300 rpm孵育另外3 h。
将细菌溶液转移到50 ml离心管中,并在3500 xg 和4°C下离心20分钟以收集细菌。
丢弃目标。
细菌颗粒重悬在850 MYU 大号知识产权裂解液用50 Myueru /毫升细菌蛋白酶抑制剂并转移到微量的Eppendorf管。细菌蛋白酶抑制剂应该制备和使用新鲜各一次。
超声处理10秒钟(不加热),在冰上静置2分钟,再重复2次。此步骤用于剪切DNA和破坏细菌。
在4°C下以17,000 xg离心30分钟。
收集收集。
注意:可以在凝胶和考马斯亮蓝上电泳,以确认蛋白质表达。


 


蛋白质纯化和激酶处理准备
〜400分装Myueru 谷胱甘肽琼脂糖(GST珠浆料)至eppendorf管。
两次洗具500 Myueru 冷PBS,离心小珠落洗涤之间在8000 XG 1分钟。
用500μlIP 裂解缓冲液洗涤一次,然后以8,000 xg的速度离心珠1分钟。
加入IP裂解缓冲液以制成50/50的珠子浆液和IP裂解缓冲液。
160传输Myueru 中该浆液进入一个新的离心管中,加入540 Myueru 知识产权裂解液IT和50 Myueru /毫升细菌蛋白酶抑制剂。细菌蛋白酶抑制剂应该制备和使用新鲜各一次。
300添加Myueru 细菌裂解物和旋转采用旋转翻滚式旋转器完以30rpm 2小时。在4 ℃下
小珠落在离心8000 XG 1个分钟,洗三次,500 Myueru IP裂解液离心小珠落洗涤之间在8000 XG 1分钟。
两次洗具500 Myueru 激酶缓冲液离心小珠落洗涤之间在8000 XG 1分钟(留出50/50浆)。 
 


激酶治疗
通过添加200μl 激酶缓冲液(400UN PKA作为干燥沉淀物)来重构PKA,并在室温下静置20分钟,这就是您的PKA解决方案。
等分试样2 Eppendorf Tubes与40μl 含上一节目的蛋白质的GST珠溶液,将一个管用作阴性对照。
加入5 Myueru pKa的解决方案要一管(分装其余部分的PKA溶液倒入5 Myueru 并储存在- 80 ℃,供将来使用)和5 Myueru 激酶解决方案(即,激酶解决方案通过无PKA)其他。请注意,PKA易冻融且在第一次冷冻后会失去一些活性,建议的等分试样量每次可用于一个治疗组,因此无需冻融.PKA冷冻> 1 后不应使用年在-80 °C
使用水浴或加热块在32 °C下孵育1小时,并每15分钟使用涡旋混合以确保珠子不会沉淀到管底部。
两次洗具500 Myueru IP裂解液缓冲液离心小珠落洗涤之间在8000 XG 1分钟,并留下在50/50浆。
30等分Myueru 解决方案到新管,并添加60 Myueru 中1.5X Laemilli 缓冲区。
煮沸5分钟,以17,000 xg离心5分钟。
转移到新鲜的微量离心管Eppendorf管中。
 


免疫印迹
将5μl 蛋白质梯子上样到15%凝胶中,然后将30μl 蛋白质样品(上一节最后一步中的上清液)上样到相邻的孔中。
用1x运行缓冲液填充PROTEAN并在200 V下运行〜45 分钟(可能会或多或少的时间,直到样品缓冲液中的蓝色染料从凝胶底部流出为止)。这些设置用于我们感兴趣的蛋白质,其他蛋白质的设置可能有所不同。
通过在100 V 下使用充满1x转移缓冲液(80%转移缓冲液,20%甲醇)的Trans-Blot将蛋白质从凝胶转移到PVDF或硝酸纤维素膜上(我们使用硝酸纤维素膜)约1.5 h(可能或多或少取决于大小蛋白质的利息)。确保凝胶和膜被放置在正确的方向,即,凝胶应该更接近于负电荷源(通常是黑色部分转移盒应该是最接近凝胶)组成,这些设置用于我们感兴趣的蛋白质,其他蛋白质的设置可能有所不同。
在感兴趣的蛋白质周围切膜(在蛋白质重量上下分别有1-2个阶梯带),并在室温下用5%BSA的TBS-T阻断30分钟,然后摇动。我们使用了一个空的显微镜盖玻片玻片盒进行孵育膜(总封闭缓冲液体积1.5米升)。一个在我们的例子,GST-Pfn1 HAS〜41的分子量的kd ,使膜切成30至70 kD的。取决于你的蛋白的感兴趣,一个的质量可能需要根据蛋白质阶梯切割不同的部分。
过夜孵育5 Pasento BSA的TBS-T以1:1000 PRRXS 底物抗体在4 。℃。与摇摆运动以10rpm使用同一容器如先前所描述,我们使用1.5M的大号阻断溶液和1.5 MYU 大号抗体。
用TBS-T清洗5分钟。
孵育随着5 Pasento BSA在TBS-T为1项小时,在室温下的以1:1 ,000抗兔过氧化物酶抗体随着摇摆。运动在10下采用相同的容器如前所述,我们用1.5米大号封闭液和1.5 MYU 大号抗体。
用T BS-T 洗涤3-4 次,每次洗涤5分钟。
清晰ECL溶液加到膜,并培育1个分钟,在室温Erature 。
膜图像(实施例结果示于图茜2,PKA治疗GST-Pfn1检测到由PRRXS 抗体,采用BioRad公司ChemiDoc )。
D:\重新格式化\ 2020-2-7 \ 1902980--1319 David Gau 837266 \图jpg \图2.jpg


2.图PRRXS 抗体检测PKA磷酸化GST-Pfn1 。GST-Pfn1被磷酸化,通过PKA和检测者PRRXS 抗体而激酶缓冲溶液处理GST-Pfn1(阴性对照)不进行检查。考马斯从这些样品也被显示了要图1示出获得磷酸带的下拉量。


 


资料分析


 


比较经过PKA处理的蛋白质样品和未经PKA处理的样品的泳道。
如果PKA处理的样品中的条带强度更高,则根据pRRXS 磷酸特异性PKA抗体,样品的磷酸化增强 (在我们的案例中,在图2中,我们发现在用PKA处理后没有谱带强度达到全谱带强度)。
 


菜谱


 


激酶缓冲液
˚F 伊纳勒集中所有组件列表如下:


20 mM BES pH 7(以粉末形式开始,在水中稀释,并在适当的pH值下稀释至合适的浓度)。它是激酶缓冲液的基础


20 mM EGTA(以粉末形式开始,添加到BES溶液中)


6 mM MgCl 2 (以粉末形式开始,添加到BES溶液中)


5 mM ATP(以粉末形式开始,添加到BES溶液中)


10 mM磷酸肌酸(以粉末形式开始,添加到BES溶液中)


1 mM DTT(以粉末形式开始,添加到BES溶液中)


IPTG解决方案
              用水和IPTG粉末制成100 mM IPTG溶液,并储存在-20 °C


 


致谢


 


这项工作得到了美国国立卫生研究院(PR)的资助(2R01CA108607)。DavidGau得到了美国国家科学基金会的博士前研究金(2012139050)和NIH心血管生物工程博士后培训资助(DG的2T32HL076124)的支持。 。


 


 


竞争利益


 


作者没有竞争利益要报告。


 


参考文献


 


豪,AK(2004)为基础的肌动蛋白调节细胞迁移到CAMP / PKA。生物化学生物生物物理学ACTA 1692(2-3):159-174。
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Copyright: © 2020 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. Gau, D. M. and Roy, P. (2020). An in vitro Assay to Screen for Substrates of PKA Using Phospho-motif-specific Antibodies . Bio-protocol 10(8): e3587. DOI: 10.21769/BioProtoc.3587.
  2. Gau, D., Veon, W., Shroff, S. G. and Roy, P. (2019). The VASP-profilin1 (Pfn1) interaction is critical for efficient cell migration and is regulated by cell-substrate adhesion in a PKA-dependent manner. J Biol Chem 294(17): 6972-6985.
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