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Substrate Specificity of Recombinant Ser/Thr Protein Kinase
重组体苏氨酸/丝氨酸蛋白激酶的底物特异性   

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Abstract

Protein kinases are enzymes that phosphorylate proteins in a cell. Determination of kinase activity in reactions of phosphorylation is a very convenient way for a biochemical characterization of this group of enzymes. Here we describe a method to determine the activity of a recombinant Ser/Thr protein kinase using as a possible substrate MBP, H1, and BSA.

Materials and Reagents

  1. Escherichia coli (E.coli) strains - for cloning and for expression - DH5α, Rosetta (DE3 pLysS)
  2. Specific synthetic oligonucleotides
  3. PCR reagents (dNTP, MgCl2, PCR buffer, Taq-polymerase, DNA) (Thermo Fisher Scientific, catalog numbers: R0181 , EP0612 )
  4. Plasmids for cloning and for expression [pTZ57R/T, pET41a(+)]
  5. CaCl2 (Sigma-Aldrich, catalog number: C3306-250G ) (for transformation)
  6. Antibiotics (Ampicillin, Kanamycine, Chloramphenicol) (AppliChem, catalog numbers: A-0839 , A-1493 , A-6435 )
  7. Isopropyl thio-β-D-galactoside (IPTG) (Fermentas, catalog number: R0393 )
  8. Tris-HCl (Sigma-Aldrich, catalog number: T6791 )
  9. Ethylenediaminetetraacetic acid (EDTA) (AppliChem, catalog number: A-1104.0500 )
  10. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815-5G )
  11. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889-10G )
  12. Na3VO4 (Sigma-Aldrich, catalog number: S6508-10G )
  13. NaF (Sigma-Aldrich, catalog number: S7920-100G )
  14. β-glycerophosphate (Sigma-Aldrich, catalog number: G9422-50G )
  15. Sucrose (AppliChem, catalog number: A-1125 )
  16. Phenylmethylsulfonyl fluoride (PMSF) (AppliChem, catalog number: A-0999 )
  17. Benzamidine hydrochloride (Sigma-Aldrich, catalog number: B6506 )
  18. 6-aminocaproic acid (Sigma-Aldrich, catalog number: A7824-25G )
  19. 2-(N-morpholino)ethanesulfonic acid (MES) (Amresco, catalog number: Am-E169-0.05 )
  20. Myelin Basic Protein bovine (MBP) (Sigma-Aldrich, catalog number: M1891-5MG )
  21. Histone from calf thymus (H1) (Sigma-Aldrich, catalog number: H5505-100MG )
  22. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470-1G )
  23. MgCl2 (AppliChem, catalog number: A-1447 )
  24. Brilliant Blue R Concentrate (Sigma-Aldrich, catalog number: B8647 )
  25. BCA Protein Assay Kit (Pierce Thermo Scientific, catalog number: 23227 )
  26. Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: L3771 )
  27. TGX FastCast Acrylamide Starter Kit (12%) (Bio-Rad Laboratories, catalog number: 161-0175 )
  28. N,N,N',N'-tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, catalog number: 161-0800 )
  29. Ammonium Persulfate (APS) (Bio-Rad Laboratories, catalog number: 161-0700 )
  30. Illustra NAP-5 Columns (GE Healthcare Life Sciences, catalog number: 17-0853-02 )
  31. [γ-32P] ATP
  32. Homogenization buffer (see Recipes)
  33. Reaction mixture (see Recipes)

Equipment

  1. Temperature controlled shaker (Heidolph, model: Unimax 1010 )
  2. French Pressure Cell Press (Aminco) or glass beads (Sigma-Aldrich, catalog number: G4649 ) for cell disruption
  3. Thermostat
  4. Spectrophotometer
  5. Gel electrophoresis equipment (Mini-PROTEAN Tetra Handcast Systems, Bio-Rad Laboratories, catalog number: 165-8000 )
  6. Laminar flow box
  7. Refrigerated microcentrifuge preset to 4 °C (Eppendorf, model: 5415 R )
  8. Gel Dryer System (Bio-Rad Laboratories, catalog number: 165-1789 )
  9. Exposure cassette/BioMax® intensifying screen set (Sigma-Aldrich, catalog numbers: C4979 and P8432 )
  10. Carestream® Kodak® autoradiography GBX developer/replenisher (Sigma-Aldrich, catalog number: P7042 )
  11. Carestream® Kodak® autoradiography GBX fixer/replenisher (Sigma-Aldrich, catalog number: P7167 )

Procedure

  1. Expression and isolation of a recombinant protein kinase
    1. Amplify a DNA fragment carrying the gene of interest using synthetic primers. For the more convenient cloning of the desired insert, it is worth to construct the primers harboring specific restriction sites for endonucleases. The easiest way to clone this PCR product is to use T-vector pTZ57R/T. Transform DH5-α strain. Spread transformed cell onto LB agar plates containing ampicillin, IPTG, X-GAL. Incubate overnight at 37 °C. Select white colonies that should harbor plasmid with a desired insert and inoculate 3 ml of LB-medium. Extract pDNA from the overnight culture. Check it for containing your full length insert either via PCR or via restriction analysis.
    2. Ligate your restriction fragment with the appropriate expression vector.
    3. Transform E. coli expression strain with the resultant plasmid using CaCl2 method (Sambrook et al., 1989). Spread transformed cells on the LB-plate containing specific antibiotic that is coded by the expression plasmid. Manipulations with bacterial cells should be carried out in a flow box (He, 2011).
    4. Grow the transformed cells in 100 ml of LB-medium until the OD595 of suspension reached 0.5. Initiate overproduction of the recombinant protein by the addition of IPTG to a final concentration of 0.1 mM. Induction conditions may vary strongly (from 3 h at 37 °C to 15 h at 25 °C). Centrifuge cells at 3,500 x g for 10 min at 4 °C.
    5. Resuspend the collected cells in a prechilled homogenization buffer (1 g of cells in up to 5 ml of buffer). Disrupt the cells either using a French Pressure Cell Press (2.8 MPa) or glass beads (use an equal volume of beads and cell suspension. Vortex vigorously for 30 sec. To avoid warming of the sample incubate a test tube for 30 sec on ice. Repeat this procedure 10 times). All procedures should be carried out at 4 °C.
    6. Centrifuge the crude extracts stepwise, first at 7,500 x g for 15 min to remove unbroken cells and afterwards at 16,000 x g for 20 min for clarifying cell lysate. Procedures should be carried out at 4 °C. Collect supernatant (soluble protein fraction) and keep a pellet.
    7. Desalt the soluble protein fraction on NAP-5 columns according manufacturer’s instruction. Elute the protein with 20 mM MES-КОН (pH 6.5) and determine protein concentration using BCA Protein Assay Kit. To determination protein quantity in the pellet resuspend it with 4% of SDS and recover the proteins by centrifugation at 16,000 x g for 20 min. Then determine protein concentration with BCA Protein Assay Kit. Mix protein sample with appropriate amount of Laemmli sample buffer to get the final protein concentration 1 mg/ml (He, 2011).
    8. Prepare and run SDS-PAGE. Stain the gel with CBB R250 (Yan, 2011).
    9. Analyze both the desalted soluble fraction and proteins from the pellet using SDS-PAGE. If your protein is soluble proceed to the protein kinase assay. If the protein is in the pellet, it is necessary to recover the protein from inclusion bodies or to start over induction procedure in other conditions, trying to shift the protein into the supernatant. Alternatively, purify the soluble proteins further by affinity chromatography. The first possibility allows to obtain the result much quickly. The second one requires more time and an additional purification step but gives the better picture with the fewer non specific bands.

  2. Phosphorylation in vitro
    1. Kinase activity assay is performed in a reaction mixture containing 20 mM MES-КОН (pH 6.5), 10 mM MgCl2, 10 µM АТP, exogenous protein substrate, 74 kBq 2μCi [γ-32P] ATP (specific activity 148 TBq/mmol). The quantity of the labeled ATP depends on the quantity of the protein. Different proteins (histone Н1, basic myelin protein МВР, and BSA) can be used as possible exogenous substrates. To calculate the volume of the [γ-32P] ATP required you can use, for example, this site http://www.radprocalculator.com/Decay.aspx. Or you can do it manually:
      Radioactive concentration (µCi/µl) =Calibration Concentration (µCi)/2 N/14.3,
      where N - number of days passed from the day of calibration, taken from the passport of the product. 14.3 days - half-life period of the 32P.
      The reaction is initiated by the addition of 5-100 µg of the recombinant protein kinase and allowed to proceed for 15 min at 30 °C. The quantity of the recombinant protein kinase in this reaction depends on whether affinity purified or non-purified the recombinant protein kinase is used.
    2. The reaction is terminated by adding half of the reaction volume of 3x Laemmli sample buffer and samples are boiled for 5 min.
    3. Phosphorylated products are resolved on SDS-polyacrylamide gels. The gels are then stained with CBB R250 as mentioned above, dried, and subjected to autoradiography. Exposition of dried gels with the X-Ray film varies from few hours at room temperature to few days at -70 °C and depends on the quantity and the activity of the recombinant protein kinase.
    4. If the recombinant protein kinase is enzymatically active it can demonstrate either autophoshorylation or phosphorylation of the substrate exogenously provided. So, it is possible to make a decision about substrate specificity of the recombinant protein kinase examined. According to the results obtained we can conclude that at the phosphorylation condition described here the examined recombinant protein kinase is capable to phosphorylate histone H1. Since at the autoradioradiograph there is no a signal corresponding to apparent molecular weight of the recombinant protein kinase it does not undergo autophosphorylation.

Representative data



Figure 1. The substrate specificity of recombinant SpkE. Coomassie R-250 staining (at the top) and autoradiographs of the corresponding gels (at the bottom). As potential substrates were examined (A) histone H1 and (B) MBP. Soluble proteins were isolated from E. coli cells with pET41a(+) (1) before and (2) after induction with IPTG and E. coli cells with pET41a(+) carrying Synechocystis spkE (3) before and (4) after induction with IPTG. About 10 μg of the E. coli cells soluble protein fraction was mixed in 25 μl with 5 μg either H1 or MBP in the presence of 1.5 mCi of [γ-32P]ATP. The phosphorylation reaction was carried out for 15 min at 30 °C. As demonstrated above only histone H1 is phosphorylated by SpkE. [modified from Zorina et al. (2014)]

Recipes

  1. Homogenization buffer
    50 mM Tris–HCl (pH 7.6)
    10 mM MgCl2
    2 mM EDTA
    1 mM DTT
    1 mM EGTA
    2 mM Na3VO4
    10 mM NaF
    50 mM β-glycerophosphate
    250 mM sucrose
    1 mM PMSF
    1 mM benzamidine hydrochloride
    0.5 mM 6-aminocaproic acid
  2. Reaction mixture (total volume 25 µl)
    20 mM MES-KOH (pH 6.5)
    10 mM MgCl2
    10 µM ATP (unlabelled)
    0.25 mg/ml MBP, or 0.5 mg/ml H1, or 1 mg/ml BSA
    2 µCi [γ-32P]ATP
    Protein kinase
    ddH2O

Acknowledgments

This work was supported by the grant from Russian Science Foundation no. 14-24-00020.

References

  1. He, F. (2011). Standard DNA cloning. Bio-protocol Bio101: e52.
  2. He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80.
  3. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
  4. Yan, Q. (2011). Rapid coomassie protein SDS-gel staining. Bio-protocol Bio101: e83.
  5. Zorina, A. A., Bedbenov, V. S., Novikova, G. V., Panichkin, V. B. and Los, D. A. (2014) Involvement of serine/threonine protein kinases in the cold stress response in the cyanobacterium Synechocystis sp. PCC 6803: Functional characterization of SpkE protein kinase. Mol Biol (Moscow) 48: 390–398.
  6. Zorina, A., Stepanchenko, N., Novikova, G. V., Sinetova, M., Panichkin, V. B., Moshkov, I. E., Zinchenko, V. V., Shestakov, S. V., Suzuki, I., Murata, N. and Los, D. A. (2011). Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the Cyanobacterium synechocystis. DNA Res 18(3): 137-151.

简介

蛋白激酶是磷酸化细胞中蛋白质的酶。 磷酸化反应中激酶活性的测定是这组酶的生物化学表征的非常方便的方法。 在这里,我们描述了一种使用可能的底物MBP,H1和BSA确定重组Ser/Thr蛋白激酶活性的方法。

材料和试剂

  1. 用于克隆和表达的大肠杆菌(
  2. 特定合成寡核苷酸
  3. PCR试剂(dNTP,MgCl 2,PCR缓冲液,Taq聚合酶,DNA)(Thermo Fisher Scientific,目录号:R0181,EP0612)
  4. 用于克隆和表达的质粒[pTZ57R/T,pET41a(+)]
  5. CaCl 2(Sigma-Aldrich,目录号:C3306-250G)(用于转化)
  6. 抗生素(氨苄青霉素,卡那霉素,氯霉素)(AppliChem,目录号:A-0839,A-1493,A-6435)
  7. 异丙基硫代-β-D-半乳糖苷(IPTG)(Fermentas,目录号:R0393)
  8. Tris-HCl(Sigma-Aldrich,目录号:T6791)
  9. 乙二胺四乙酸(EDTA)(AppliChem,目录号:A-1104.0500)
  10. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815-5G)
  11. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889-10G)
  12. Na 3 O 4(Sigma-Aldrich,目录号:S6508-10G)
  13. NaF(Sigma-Aldrich,目录号:S7920-100G)
  14. β-磷酸甘油酯(Sigma-Aldrich,目录号:G9422-50G)
  15. 蔗糖(AppliChem,目录号:A-1125)
  16. 苯基甲基磺酰氟(PMSF)(AppliChem,目录号:A-0999)
  17. 苄脒盐酸盐(Sigma-Aldrich,目录号:B6506)
  18. 6-氨基己酸(Sigma-Aldrich,目录号:A7824-25G)
  19. 2-(N-吗啉代)乙磺酸(MES)(Amresco,目录号:Am-E169-0.05)
  20. 髓鞘碱性蛋白牛(MBP)(Sigma-Aldrich,目录号:M1891-5MG)
  21. 来自小牛胸腺的组蛋白(H1)(Sigma-Aldrich,目录号:H5505-100MG)
  22. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:05470-1G)
  23. MgCl 2(AppliChem,目录号:A-1447)
  24. Brilliant Blue R Concentrate(Sigma-Aldrich,目录号:B8647)
  25. BCA蛋白测定试剂盒(Pierce Thermo Scientific,目录号:23227)
  26. 十二烷基硫酸钠,SDS(Sigma-Aldrich,目录号:L3771)
  27. TGX FastCast丙烯酰胺起始试剂盒(12%)(Bio-Rad Laboratories,目录号:161-0175)
  28. N,N,N',N'-四甲基乙二胺(TEMED)(Bio-Rad Laboratories,目录号:161-0800)
  29. 过硫酸铵(APS)(Bio-Rad Laboratories,目录号:161-0700)
  30. Illustra NAP-5柱(GE Healthcare Life Sciences,目录号:17-0853-02)
  31. [γ- 32 P] ATP
  32. 均质缓冲液(参见配方)
  33. 反应混合物(参见配方)

设备

  1. 温度控制振荡器(Heidolph,型号:Unimax 1010)
  2. French Amino Cell Press(Aminco)或玻璃珠(Sigma-Aldrich,目录号:G4649)用于细胞破碎
  3. 恒温器
  4. 分光光度计
  5. 凝胶电泳装置(Mini-PROTEAN Tetra Handcast Systems,Bio-Rad Laboratories,目录号:165-8000)
  6. 层流箱
  7. 冷藏微量离心机预设为4°C(Eppendorf,型号:5415 R)
  8. 凝胶干燥器系统(Bio-Rad Laboratories,目录号:165-1789)
  9. 曝光盒/BioMax增强屏幕组(Sigma-Aldrich,目录号:C4979和P8432)
  10. Carestream Kodak 放射自显影GBX显影剂/补充剂(Sigma-Aldrich,目录号:P7042)
  11. Carestream Kodak 放射自显影GBX定色剂/补充剂(Sigma-Aldrich,目录号:P7167)

程序

  1. 重组蛋白激酶的表达和分离
    1. 使用合成扩增携带目标基因的DNA片段 引物。 为了更方便地克隆所需的插入片段,它是 值得构建含有特异性限制性位点的引物 核酸内切酶。克隆该PCR产物的最简单的方法是使用 T载体pTZ57R/T。转化DH5-α菌株。传播转化细胞 含有氨苄青霉素,IPTG,X-GAL的LB琼脂平板上。在37℃孵育过夜  37℃。选择应含有所需质粒的白色菌落 插入并接种3ml LB-培养基。从过夜提取pDNA 文化。通过PCR检查其中是否含有全长插入片段 或通过限制性分析。
    2. 用适当的表达载体连接您的限制片段。
    3. 转换 E。大肠杆菌表达菌株与所得质粒  CaCl 2方法(Sambrook等人,1989)。传播转化细胞上 含有通过表达式编码的特异性抗生素的LB平板 质粒。操作细菌细胞应在a 流动箱(He,2011)。
    4. 生长转化的细胞在100毫升 LB-培养基中,直至悬浮液的OD 595达到0.5。 发起 通过向IPTG中加入IPTG来过量产生重组蛋白 终浓度为0.1mM。 感应条件可能变化很大 (在37℃下3小时至25℃下15小时)。 离心细胞在3,500×g 4℃下10分钟。
    5. 将收集的细胞重悬在预冷的 匀浆缓冲液(1g细胞在高达5ml缓冲液中)。 中断 使用French Pressure Cell Press(2.8MPa)或玻璃 珠(使用等体积的珠和细胞悬浮液 剧烈30秒。 为了避免样品升温孵化一个测试 管在冰上30秒。 重复此过程10次)。 所有程序 应在4℃下进行
    6. 离心粗提取物 逐步,首先在7,500×g 下15分钟以除去未破碎的细胞和 然后以16,000×g离心20分钟以澄清细胞裂解物。 程序应在4°C进行。 收集上清液(可溶 蛋白质级分)并保持沉淀。
    7. 去除可溶性蛋白 部分在NAP-5柱上。 洗脱 该蛋白用20mM MES-КОН(pH6.5)并测定蛋白质 使用BCA蛋白测定试剂盒。 测定蛋白 在沉淀中的量用4%SDS重悬并回收 蛋白质通过在16,000×g离心20分钟。 然后确定 蛋白浓度与BCA蛋白测定试剂盒。 混合蛋白样品 用适量的Laemmli样品缓冲液得到最终 蛋白质浓度1mg/ml(He,2011)
    8. 准备并运行SDS-PAGE。 用CBB R250染色凝胶(Yan,2011)。
    9. 分析脱盐的可溶性级分和蛋白质 使用SDS-PAGE沉淀。 如果你的蛋白质是可溶的,继续到蛋白质   激酶测定。 如果蛋白质在沉淀中,则有必要 从包涵体中回收蛋白质或开始过感应 程序在其他条件下,试图将蛋白质转移到 上清液。 或者,通过进一步纯化可溶性蛋白质 亲和色谱法。 第一种可能性允许获得 结果很快。 第二个需要更多的时间和额外的   纯化步骤,但给出更少的非更好的图片 特定频带

  2. 体外磷酸化
    1. 激酶活性测定在含有20μg/ mM MES-КОН(pH 6.5),10mM MgCl 2,10μMATP,外源蛋白 底物,74kBq2μCi[γ-32 P] ATP(比活性148TBq/mmol)。的  标记的ATP的量取决于蛋白质的量。 不同的蛋白质(组蛋白Н1,碱性髓鞘蛋白МВР和BSA)可以 用作可能的外源基质。要计算的音量  [γ- 32 P] ATP需要您可以使用,例如,本网站 http://www.radprocalculator.com/Decay.aspx。或者您可以手动进行:
      放射性浓度(μCi/μl)=校准浓度(μCi)/2 N/14.3 ,
      其中N - 从校准当天起的天数,取自  产品的护照。 14.3天 - P的半衰期。
      通过加入5-100μg的β-葡聚糖引发反应 重组蛋白激酶,并使其在30℃下进行15分钟。 该反应中重组蛋白激酶的量取决于 关于是否亲和纯化或未纯化重组蛋白 激酶。
    2. 通过加入一半的反应终止反应 反应体积为3×Laemmli样品缓冲液,样品煮沸5分钟   min。
    3. 磷酸化产物在SDS-聚丙烯酰胺上分离   凝胶。 然后如上所述用CBB R250染色凝胶, 干燥,并进行放射自显影。 干凝胶的展览 X射线膜在室温下几小时至几天内变化 -70℃,并且取决于重组体的量和活性 蛋白激酶
    4. 如果重组蛋白激酶是 酶活性,它可以显示自磷酸化或 磷酸化外源提供的底物。 所以,它是 可能做出关于底物特异性的决定 重组蛋白激酶检测。 根据获得的结果 我们可以得出结论,在这里描述的磷酸化条件   检查的重组蛋白激酶能够磷酸化组蛋白 H1。由于在自动放射自显影仪上没有相应的信号  到其所表达的重组蛋白激酶的表观分子量 不进行自磷酸化。

代表数据



图1.重组SpkE的底物特异性。 考马斯R-250染色(在顶部)和相应凝胶的放射自显影(在底部)。作为潜在底物检查(A)组蛋白H1和(B)MBP。从E中分离可溶性蛋白质。在用IPTG和EM诱导前和(2)后用pET41a(+)(1)诱导大肠杆菌细胞。在用IPTG诱导前和(4)后用携带集胞藻spkE(3)的pET41a(+)的大肠杆菌细胞。约10μg的E。在1.5mCi的[γ-32P] ATP存在下,将大肠杆菌细胞可溶性蛋白级分在25μl中与5μgH1或MBP混合。磷酸化反应在30℃下进行15分钟。如上所述,只有组蛋白H1被SpkE磷酸化。 [修改自Zorina等人(2014)]

食谱

  1. 均匀化缓冲液
    50mM Tris-HCl(pH7.6)
    10mM MgCl 2/
    2mM EDTA 1 mM DTT
    1 mM EGTA
    2mM Na 3+ VO 4
    10mM NaF 50 mMβ-甘油磷酸盐 250mM蔗糖 1mM PMSF
    1mM苄脒盐酸盐 0.5mM 6-氨基己酸
  2. 反应混合物(总体积25μl)
    20mM MES-KOH(pH 6.5)
    10mM MgCl 2/
    10μMATP(未标记)
    0.25mg/ml MBP或0.5mg/ml H1或1mg/ml BSA 2μCi[γ- 32 P] ATP
    蛋白激酶
    ddH sub 2 O

致谢

这项工作得到了俄罗斯科学基金会的资助。 14-24-00020。

参考文献

  1. 他,F.(2011)。 标准DNA克隆 生物协议 Bio101:e52。
  2. 他,F.(2011)。 Laemmli-SDS-PAGE。生物协议 Bio101:e80。
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引用:Zorina, A. A., Novikova, G. V. and Los, D. A. (2015). Substrate Specificity of Recombinant Ser/Thr Protein Kinase. Bio-protocol 5(6): e1426. DOI: 10.21769/BioProtoc.1426.
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