Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis

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Molecular Cell
Oct 2018



Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling by "click" chemistry. Here we describe a protocol to measure protein synthesis by AHA labeling and flow cytometry. Taking advantage of gating different cell populations, we provide a typical example of the flow cytometric-based analysis of protein synthesis during the cell cycle. While we used mouse B cells in this protocol this method can be readily applied to any cell types and organisms.

Keywords: Protein synthesis (蛋白合成), Translation (翻译), AHA (叠氮丙氨酸), Click chemistry (点击化学), Flow cytometry (流式细胞术), Cell cycle (细胞周期), Mitosis (有丝分裂), Non-radioactive amino acid (非放射性氨基酸)


Traditionally measurement of protein synthesis is performed by pulse labeling of translation products using radiolabeled amino acids such as [35S]methionine and [35S]cysteine but the use of radioactive materials is the major disadvantage. Recent advance in bio-orthogonal chemical reporters such as azides and alkynes allows us to quantitatively monitor and track biomolecules (proteins, lipids or nucleic acids) through click chemistry (Dieterich, 2010). Azidohomoalaine (AHA) (Figure 1) is a methionine analog that contains an azide moiety and is incorporated into newly synthesized proteins. Thus, nascent proteins can be detected through "click" reaction between AHA (azido) and a fluorescent alkyne (Dieterich, 2010).

Figure 1. Chemical structures of L-methionine and L-azidohomoalaine (AHA)

Measuring protein synthesis during the cell cycle in mammalian cells has been challenging. To do this, cell synchronization in specific phases of the cell cycle is necessary but the use of drugs such as nocodazole was shown to affect translation (Coldwell et al., 2013; Shuda et al., 2015). To measure protein synthesis without drug treatment, here we used metabolic pulse labeling with AHA in a flow cytometric assay (Kiick et al., 2002; Shuda et al., 2015). In combination with AHA, we used anti-phospho-Histone H3 (Ser10) antibody as a mitotic marker to monitor mitotic translation in mouse B lymphoma cells (19DN) (Sander et al., 2015).

Materials and Reagents

  1. Pipette tips (Eppendorf, epT.I.P.S.)
  2. 6-well plate (Thermo Fisher Scientific, BioLite 6-well Multidish, catalog number: 130184)
  3. 96-well U- or V-bottom cell culture plate (Corning, catalog number: 6902D09 or 6928A17)
  4. 5 ml round bottom polystyrene FACS tube (Falcon, catalog number: 38007)
  5. L-Azidohomoalanine (AHA) (Anaspec, catalog number: AS-63669, 500 mM stock solution in deionized water, keep at -20 °C up to at least a few months)
  6. Methionine-free medium such as Dulbecco’s Modified Eagle’s Medium–high glucose With 4,500 mg/L glucose and sodium bicarbonate, without L-methionine, L-cystine and L-glutamine, liquid, sterile-filtered, suitable for cell culture (Merk, catalog number: D0422)
  7. Fetal bovine serum, South America origin, dialyzed, sterile filtered (Pan Biotech, catalog number: P30-2102)
  8. L-Methionine (Merck, catalog number: M9625, make 300 mM stock solution in deionized water, keep at -20 °C up to at least a few months)
  9. L-Cystine dihydrochloride (Merck, catalog number: C2526, prepare fresh 200 mM (1,000x for DMEM) solution in deionized water)
  10. GlutaMAX (Thermo Fisher Scientific, catalog number: 35050061, 200 mM stock solution)
  11. D-PBS (1x), no calcium chloride, no magnesium chloride, sterile-filtered (Thermo Scientific Fisher, catalog number: 14190144) 
  12. Alexa Fluor488 alkyne (Thermo Fisher Scientific, catalog number: A10267)
  13. Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor 647 Conjugate) (Cell Signaling Technology, catalog number: 3458S) 
  14. (+)-Sodium L-ascorbate (Merck, catalog number: A7631) 
  15. Copper(II)-Sulphate (CuSO4) (Baseclick, catalog number: BCMI-004-50) 
  16. Paraformaldehyde (PFA) (Roth, 335)
  17. Saponin (Sigma-Aldrich, catalog number: 47036)
  18. (Optional) Triton X-100 (Sigma-Aldrich, catalog number: T9284) 
  19. Bovine serum albumin (BSA) (Roth, 8076)
  20. DMSO (Merck, catalog number: D8418)
  21. EDTA (Merck, catalog number: EDS)
  22. NaN3 (Merck, catalog number: 71289)
  23. Cycloheximide (Santa Cruz Biotechnology, catalog number: sc-3508)
  24. Cell culture medium for AHA labeling (w/o methionine or AHA) (see Recipes)
  25. Washing solution (see Recipes)
  26. Fixing solution (see Recipes)
  27. Permeabilization solution (see Recipes)
  28. FACS buffer (see Recipes)


  1. (Optional) Multi channel pipette (Eppendorf, catalog number: 3122 000.043 [10-100 μl])
  2. CO2 Incubator (Thermo Fisher Scientific, catalog number: 51026331)
  3. Refrigerated centrifuge (Eppendorf, models: 5418 R [fixed angle for tubes], 5804 R [swing out for 96 well plates])
  4. Flow cytometer (FACS Aria instrument) (BD Biosciences)


  1. Analysis Software: FlowJo


  1. AHA pulse labeling
    1. Seed cells in a 6-well plate containing normal cell culture medium so that cells should be 50%-60% confluent next day. Mouse B cells (in suspension) were used in this study but any cell types can be used.
    2. Next day, starve cells in the methionine-free medium for 30 min.
      Note: This step may be omitted to avoid cellular stress.
    3. Start pulse labeling by adding 1 mM AHA or methionine (negative control) to the methionine-free medium and incubate cells for at least 10 min in a 37 °C CO2 Incubator. Cycloheximide treatment (100 μg/ml) can be used as a negative control as well.
    4. Wash cells with ice-cold PBS. Spin down cells at 500 x g, 4 °C for 3 min and remove the supernatant. Repeat this step once more. For adherent cells such as HEK293, 1 mM EDTA in PBS can be used to harvest cells.
      Note: From this step, we used 96-well U- or V-bottom cell culture plate for subsequent steps to increase the throughput. For subsequent steps, use 100-200 μl solution to suspend cells after spinning.
    5. Fix cells in fixing solution (4% PFA in PBS) for 15 min at RT. Spin down cells and remove the supernatant.
    6. Permeabilize cells with 1% BSA with 0.2% saponin (or 0.25% Triton X-100) in PBS for 15 min. Spin down cells at 500 x g, at RT for 3 min and remove the supernatant.

  2. Click reaction
    1. Prepare the click reaction solution (see Recipe 5).
    2. Incubate samples with 100 μl of the click solution for 30 min at RT. Protect from light.
    3. Wash cells once with 1% BSA with 0.2% saponin in PBS. Spin down cells and remove the supernatant. In case of Trition X-100 is used for permeabilization, wash cells with 1% BSA in PBS.

  3. Immunolabeling of a mitotic marker, histone H3 phospho Ser10
    1. Incubate samples with anti-phospho histone pH3 antibody (1:100, v/v) in 100 μl 1% BSA in PBS for 60 min. Protect from light. 
    2. Wash cells twice with 1% BSA in PBS. Spin down cells and remove the supernatant.
    3. Resuspend in FACS buffer (1% FBS, 1 mM EDTA, 0.05% NaN3 in PBS) and transfer cells into a 5 ml round bottom polystyrene FACS tube.

  4. Flow cytometric analysis
    1. Gate the main cell population by the forward (FSC-A) and side scatter profile (SSC-A) to exclude cellular debris and dead cells (Figure 2A). Gate further the singlet subset by SSC-H and SSC-W (Figure 2B).
    2. For the subset of cells gated in the Step D1, record AHA-incorporation (y-axis, alexa488) and the mitotic marker (x-axis, alexa647) for at least 10,000 events (Figure 2C). Quantify AHA intensities (y-axis) in interphase and mitotic cells (x-axis) that are separated based on fluorescence intensity of the mitotic marker (see Step D4).
      Note: Prepare single color (stained with the Alexa Fluor488-AHA or the Alexa Fluor 647 conjugated antibody) and no color controls for setting FACS parameters and compensation on the flow cytometer. Each compensation control is acquired on the flow cytometer to determine compensation values for each fluorochrome combination. This protocol does not cover detailed instructions for FACS parameter setup. For assistance, please contact your FACS core facility.
    3. Check if the negative control sample (methionine-labeled cells) shows the low level of AHA-incorporation (Figure 2D). 
    4. Quantify the AHA intensities for individual cells (e.g., median intensity) in interphase and mitosis using FlowJo. To do this, open FlowJo and load the files (.fcs) into a workspace. Double-click on a file in the workspace and a plot like Figure 2A will appear. Select subsets for further analyses based on the forward (FSC-A) and side scatter profile (SSC-A) as described in Step D1. Select corresponding populations of interphase and mitosis (see Figure 2C) and click on “Workspace” → “Statistics” → “Median” → “AHA”, which calculates median intensities of AHA signal. Examples of data analysis are shown in Figure 2E.

      Figure 2. Monitoring global protein synthesis in interphase and mitosis. A and B. Gating strategy. C. The main cell population was further analyzed by dual staining for phospho-H3 S10 and AHA-labeled proteins. Global protein synthesis was monitored by AHA incorporation (y-axis) in interphase and mitosis (based on H3 pS10 staining, x-axis). D. As a negative control, methionine incorporation into proteins instead of AHA was also monitored. E. Examples of quantitative outputs: each dot represents the median AHA intensity calculated from the interphase or mitotic cell population in an independent experiment. The results from three independent experiments are shown.


  1. Cell culture medium for AHA labeling (w/o methionine or AHA)
    500 ml of DMEM without methionine, cysteine, glutamine
    50 ml of dialyzed serum (normal serum can be used in case use of dialyzed serum affects cell growth.)
    1. As long as methionine-free medium is used, the use of normal serum instead of dialyzed serum will not significantly affect pulse labeling results.
    2. For Cysteine and GlutaMAX, follow the defined concentration of the medium you use.
    3. Directly add methionine or AHA to the medium just before pulse labeling.
  2. Washing solution
    1% BSA in PBS 
  3. Fixing solution
    4% PFA in PBS
  4. Permeabilization solution
    1% BSA with 0.2% saponin (or 0.25% Triton X-100) in PBS 
  5. Click reaction solution

  6. FACS buffer
    1% FBS
    1 mM EDTA
    0.05% NaN3 in PBS


You can perform washing, fixing, click reaction and etc. in a 96-well U- or V-bottom cell culture plate to increase the throughput. You can sort the AHA-labeled cells and the sorted cells can be used for many applications including western blot analysis and quantitative analysis using mass spectrometry.


We would like to thank Shuda et al. for describing the original protocol (Shuda et al., 2015). We adapted and modified the protocol for this study. We would also like to thank Matthias Selbach (Max Delbruck Center for Molecular Medicine) for critical reading of the manuscript and for his advice.

Competing interests

The authors declare no conflicts of interest or competing interests.


  1. Coldwell, M. J., Cowan, J. L., Vlasak, M., Mead, A., Willett, M., Perry, L. S. and Morley, S. J. (2013). Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis. Cell Cycle 12(23): 3615-3628.
  2. Dieterich, D. C. (2010). Chemical reporters for the illumination of protein and cell dynamics. Curr Opin Neurobiol 20(5): 623-630.
  3. Kiick, K. L., Saxon, E., Tirrell, D. A. and Bertozzi, C. R. (2002). Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci U S A 99(1): 19-24.
  4. Shuda, M., Velasquez, C., Cheng, E., Cordek, D. G., Kwun, H. J., Chang, Y. and Moore, P. S. (2015). CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation. Proc Natl Acad Sci U S A 112(19): 5875-5882.
  5. Sander, S., Calado, D.P., Srinivasan, L., Ko¨ chert, K., Zhang, B., Rosolowski, M., Rodig, S.J., Holzmann, K., Stilgenbauer, S., Siebert, R., Bullinger L., Rajewsky K. (2012). Synergy between PI3K signaling and MYC in Burkitt lymphomagenesis. Cancer Cell 22: 167-179.


蛋白质合成是维持细胞蛋白质稳定的最基本的生物学过程之一。 叠氮高丙氨酸(AHA)是甲硫氨酸的非放射性和“可点击的”氨基酸类似物,其可以掺入新合成的蛋白质中。 因此,可以通过“点击”化学的荧光标记来检测和定量AHA标记的新生蛋白质。 在这里,我们描述了通过AHA标记和流式细胞术测量蛋白质合成的方案。 利用门控不同的细胞群,我们提供了细胞周期中基于流式细胞仪的蛋白质合成分析的典型实例。 虽然我们在该方案中使用了小鼠B细胞,但该方法可以容易地应用于任何细胞类型和生物体。
【背景】传统上,蛋白质合成的测量是通过使用放射性标记的氨基酸如[ 35 S]甲硫氨酸和[ 35 S]半胱氨酸对翻译产物进行脉冲标记来进行的,但使用放射性物质是主要的缺点。生物正交化学报道分子如叠氮化物和炔烃的最新进展使我们能够通过点击化学定量监测和跟踪生物分子(蛋白质,脂质或核酸)(Dieterich,2010)。叠氮基高粱碱(AHA)(图1)是含有叠氮部分的甲硫氨酸类似物,并掺入新合成的蛋白质中。因此,可以通过AHA(叠氮基)和荧光炔烃之间的“点击”反应来检测新生蛋白质(Dieterich,2010)。

图1. L-蛋氨酸和L-叠氮基高

在哺乳动物细胞的细胞周期中测量蛋白质合成具有挑战性。要做到这一点,细胞周期的特定阶段的细胞同步是必要的,但是使用诸如诺考达唑的药物会影响翻译(Coldwell et al。,2013; Shuda et al。 ,2015)。为了在没有药物治疗的情况下测量蛋白质合成,这里我们在流式细胞术测定中使用AHA代谢脉冲标记(Kiick et al。,2002; Shuda et al。,2015) 。与AHA组合,我们使用抗磷酸组蛋白H3(Ser10)抗体作为有丝分裂标记物来监测小鼠B淋巴瘤细胞中的有丝分裂翻译(19DN)(Sander 等人,2015)。

关键字:蛋白合成, 翻译, 叠氮丙氨酸, 点击化学, 流式细胞术, 细胞周期, 有丝分裂, 非放射性氨基酸


  1. 移液器吸头(Eppendorf,epT.I.P.S。)
  2. 6孔板(Thermo Fisher Scientific,BioLite 6-well Multidish,目录号:130184)
  3. 96孔U型或V型底细胞培养板(Corning,目录号:6902D09或6928A17)
  4. 5毫升圆底聚苯乙烯FACS管(Falcon,目录号:38007)
  5. L-叠氮基高丙氨酸(AHA)(Anaspec,目录号:AS-63669,在去离子水中的500 mM储备溶液,保持在-20°C至少几个月)
  6. 不含甲硫氨酸的培养基,如Dulbecco's Modified Eagle's中高葡萄糖含4,500 mg / L葡萄糖和碳酸氢钠,不含L-蛋氨酸,L-胱氨酸和L-谷氨酰胺,液体,无菌过滤,适合细胞培养(Merk,目录编号:D0422)
  7. 胎牛血清,来自南美洲,透析,无菌过滤(Pan Biotech,目录号:P30-2102)
  8. L-蛋氨酸(默克,目录号:M9625,在去离子水中制备300 mM储备液,保持在-20°C至少几个月)
  9. L-胱氨酸二盐酸盐(Merck,目录号:C2526,在去离子水中制备新鲜的200mM(1,000x用于DMEM)溶液)
  10. GlutaMAX(Thermo Fisher Scientific,目录号:35050061,200 mM储备液)
  11. D-PBS(1x),无氯化钙,无氯化镁,无菌过滤(Thermo Scientific Fisher,目录号:14190144) 
  12. Alexa Fluor488炔烃(赛默飞世尔科技,目录号:A10267)
  13. 磷酸组蛋白H3(Ser10)(D2C8)XP Rabbit mAb(Alexa Fluor 647 Conjugate)(Cell Signaling Technology,目录号:3458S) 
  14. (+) - L-抗坏血酸钠(默克,目录号:A7631) 
  15. 铜(II) - 硫酸盐(CuSO 4 )(Baseclick,目录号:BCMI-004-50) 
  16. 多聚甲醛(PFA)(Roth,335)
  17. 皂甙(西格玛奥德里奇,目录号:47036)
  18. (可选)Triton X-100(Sigma-Aldrich,目录号:T9284) 
  19. 牛血清白蛋白(BSA)(Roth,8076)
  20. DMSO(默克,目录号:D8418)
  21. EDTA(默克,目录号:EDS)
  22. NaN 3 (默克,目录号:71289)
  23. 环己酰亚胺(Santa Cruz Biotechnology,目录号:sc-3508)
  24. 用于AHA标记的细胞培养基(不含蛋氨酸或AHA)(参见食谱)
  25. 洗涤液(见食谱)
  26. 修复解决方案(参见食谱)
  27. 渗透解决方案(见食谱)
  28. FACS缓冲液(见食谱)


  1. (可选)多通道移液器(Eppendorf,目录号:3122 000.043 [10-100μl])
  2. CO 2 培养箱(Thermo Fisher Scientific,目录号:51026331)
  3. 冷冻离心机(Eppendorf,型号:5418 R [管的固定角度],5804 R [96孔板的摆动])
  4. 流式细胞仪(FACS Aria仪器)(BD Biosciences)


  1. 分析软件:FlowJo


  1. AHA脉冲标记
    1. 在含有正常细胞培养基的6孔板中培养细胞,使细胞第二天达到50%-60%汇合。在该研究中使用小鼠B细胞(悬浮液),但可以使用任何细胞类型。
    2. 第二天,在不含蛋氨酸的培养基中饥饿细胞30分钟。
    3. 通过向不含蛋氨酸的培养基中加入1mM AHA或甲硫氨酸(阴性对照)开始脉冲标记,并在37℃CO 2 培养箱中孵育细胞至少10分钟。环己酰亚胺处理(100μg/ ml)也可用作阴性对照。
    4. 用冰冷的PBS洗涤细胞。将细胞在500 x g ,4℃下离心3分钟并除去上清液。再一次重复此步骤。对于粘附细胞如HEK293,PBS中的1mM EDTA可用于收获细胞。
    5. 将细胞固定在固定溶液(PBS中的4%PFA)中,在室温下保持15分钟。旋转细胞并除去上清液。
    6. 用含有0.2%皂苷(或0.25%Triton X-100)的1%BSA在PBS中渗透细胞15分钟。将细胞在500 x g 下旋转,在室温下旋转3分钟并除去上清液。

  2. 点击反应
    1. 准备点击反应溶液(参见配方5)。
    2. 在室温下用100μl点击溶液孵育样品30分钟。避光。
    3. 用含有0.2%皂苷的PBS中的1%BSA洗涤细胞一次。旋转细胞并除去上清液。如果Trition X-100用于透化,用PBS中的1%BSA洗涤细胞。

  3. 免疫标记有丝分裂标记物,组蛋白H3磷酸Ser10
    1. 将抗体磷酸化组蛋白pH3抗体(1:100,v / v)的样品在PBS中的100μl1%BSA中孵育60分钟。避光。 
    2. 用PBS中的1%BSA洗涤细胞两次。旋转细胞并除去上清液。
    3. 重悬于FACS缓冲液(1%FBS,1mM EDTA,0.05%NaN 3 的PBS溶液)中,并将细胞转移到5ml圆底聚苯乙烯FACS管中。

  4. 流式细胞术分析
    1. 通过前向(FSC-A)和侧向散射谱(SSC-A)对主要细胞群进行门控,以排除细胞碎片和死细胞(图2A)。通过SSC-H和SSC-W进一步对单重态子集进行门控(图2B)。
    2. 对于步骤D1中门控的细胞子集,记录至少10,000个事件的AHA掺入(y轴,alexa488)和有丝分裂标记(x轴,alexa647)(图2C)。定量基于有丝分裂标记物的荧光强度分离的间期和有丝分裂细胞(x轴)中的AHA强度(y轴)(参见步骤D4)。
      注意:准备单色(用Alexa Fluor488-AHA或Alexa Fluor 647共轭抗体染色),并且没有用于在流式细胞仪上设置FACS参数和补偿的颜色控制。在流式细胞仪上获取每个补偿控制以确定每种荧光染料组合的补偿值。该协议不包括FACS参数设置的详细说明。如需帮助,请联系您的FACS核心设施。
    3. 检查阴性对照样品(蛋氨酸标记的细胞)是否显示低水平的AHA掺入(图2D)。 
    4. 使用FlowJo量化间期和有丝分裂中单个细胞的AHA强度(例如,中值强度)。为此,请打开FlowJo并将文件(.fcs)加载到工作区中。双击工作区中的文件,将出现如图2A所示的图。如步骤D1所述,根据前向(FSC-A)和侧向散射轮廓(SSC-A)选择子集以进行进一步分析。选择相应的间期和有丝分裂群体(见图2C)并点击“工作空间”→“统计”→“中位数”→“AHA”,它计算AHA信号的中值强度。数据分析的例子如图2E所示。

      图2.监测间期和有丝分裂中的全球蛋白质合成。 A和B.门控策略。 C.通过磷酸-H3 S10和AHA-标记的蛋白质的双重染色进一步分析主要细胞群。通过间期和有丝分裂中的AHA掺入(y轴)监测全局蛋白质合成(基于H3 pS10染色,x轴)。 D.作为阴性对照,还监测蛋氨酸掺入蛋白质而不是AHA。 E.定量输出的实例:每个点代表在独立实验中从间期或有丝分裂细胞群计算的中值AHA强度。显示了三个独立实验的结果。


  1. 用于AHA标记的细胞培养基(不含蛋氨酸或AHA)
    1. 只要使用不含蛋氨酸的培养基,使用正常血清代替透析血清不会显着影响脉冲标记结果。
    2. 对于半胱氨酸和GlutaMAX,请遵循您使用的介质的定义浓度。
    3. 在脉冲标记之前直接将甲硫氨酸或AHA添加到培养基中。
  2. 洗涤液
  3. 修复解决方案
  4. 透化解决方案
    PBS中含有0.2%皂苷(或0.25%Triton X-100)的1%BSA 
  5. 点击反应解决方案

  6. FACS缓冲区
    1 mM EDTA
    PBS中0.05%NaN 3




我们要感谢Shuda et al。描述原始协议(Shuda et al。,2015)。我们修改并修改了本研究的方案。我们还要感谢Matthias Selbach(Max Delbruck分子医学中心)对手稿的批判性阅读和他的建议。




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引用:Imami, K. and Yasuda, T. (2019). Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis. Bio-protocol 9(8): e3215. DOI: 10.21769/BioProtoc.3215.