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Assessment of Modulation of Protein Stability Using Pulse-chase Method
使用脉冲追踪法评估蛋白质稳定性的调节   

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Jia Li
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Abstract

Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows quantitative analysis of modulation of protein or mRNA stability under different treatments or culturing conditions.

Keywords: Pulse-chase(脉冲追踪), Protein stability(蛋白质稳定性), Half-life(半衰期), Protein degradation(蛋白质降解), Proteasome(蛋白酶体), Lysosome(溶酶体), MCL-1(MCL-1), mTOR(mTOR), Sunitinib(舒尼替尼)

Background

Pulse-chase technique is a method that involves culturing cells in a medium containing labeled amino acids for a short period of time known as ‘pulse’. This results in the generation of newly-synthesized polypeptides incorporating labeled amino acids. The pulse step is followed by a ‘chase’ step in which labeling medium is washed out to terminate the labeling process and is replaced by a medium with non-labeled amino acids to allow for the quantification of the initially synthesized radiolabeled proteins at any given time during this chase phase. This technique thus allows quantitative analysis of the processing of a protein of interest from synthesis to degradation in a timely fashion. Pulse-chase method can be used to analyze a variety of processes such as protein folding, co-translational modifications and intracellular transport (Jansens and Braakman 2003; Magadán, 2014). However, pulse-chase technique is most commonly used to assess the stability of a protein under different experimental conditions.

Radioactivity is a commonly used label. Labeling is usually done for proteins using radioactive S35 Methionine. The kinetics of disappearance of the radiolabeled proteins relies on how fast these proteins get degraded which can be exploited to examine the effect of different experimental conditions on protein stability. To assess the stability of a particular protein of interest, this protein is immunoprecipitated from all other immunolabeled proteins that were produced during the pulse period using a specific antibody. Immunoprecipitation will isolate a ratio of radiolabeled protein that was produced during the pulse and has not been degraded until the time of immunoprecipitation. Then radioactivity is measured to assess the relative amounts of labeled protein over a period of time.

While the focus of this protocol is the assessment of protein stability, pulse-chase method can also be used to assess the stability of mRNA. Labeling of mRNA during the pulse step can be done using 3UTP. Quantitative analysis of mRNA of interest then follows by taking aliquots of total mRNA at different time points and applying one of two methods: 1) Dot blot method in which the labeled mRNA of interest anneals to a complementary single stranded nucleic acid attached to a filter paper, other labeled mRNA are then washed out and radioactivity is measured by autoradiogram or scintillation counting, 2) Affinity purification method in which the labeled mRNA of interest anneals to a complementary anchored RNA/DNA immobilized to beads, the beads are then collected by centrifugation and radioactivity of the captured labeled mRNA is quantified.

Materials and Reagents

Other than the materials routinely used for mainlining cells in tissue culture and for immunoprecipitation and SDS-PAGE, the specific reagents required for the pulse-chase experiment are:

  1. Pipette tips
  2. 1.5 ml micro-centrifuge tubes
  3. Cell culture dishes
  4. Plastic cell scraper
  5. Tissue culture medium, appropriate for the cell line used, without methionine and cysteine
  6. DPBS
  7. Dithiothreitol (DTT)
  8. Protease inhibitor cocktail
  9. Phosphatase inhibitor cocktail (Sigma-Aldrich, catalog number: P2850 )
  10. N-Ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: R3876 )
    Note: This product has been discontinued.
  11. Bradford colorimetric assay
  12. Protein A-Sepharose bead
  13. [35S]-Methionine (PerkinElmer, catalog number: NEG709A500UC )
  14. Methionine (250 mM in H2O, store at -20 °C) (Sigma-Aldrich, catalog number: M5308 )
  15. Cysteine (500 mM in H2O, store at -20 °C) (Sigma-Aldrich, catalog number: C7352 )
  16. Sodium chloride (NaCl)
  17. Triton X-100
  18. Ethylenediaminetetraacetic acid (EDTA)
  19. Glycerol
  20. Tris-HCl
  21. Sodium dodecyl sulfate (SDS)
  22. Methanol
  23. Acetic acid
  24. Pulse (labeling) medium (see Recipes)
  25. Chase medium (see Recipes)
  26. Lysis buffer (see Recipes)
  27. Wash buffer (see Recipes)
  28. Fixation solution (see Recipes)

Equipment

  1. Pipettes
  2. Refrigerated centrifuge
  3. 95 °C heat block
  4. Cell culture incubator (37 °C humidified 5% CO2)
  5. SDS-PAGE apparatus
  6. Radiographic cassette
  7. -80 °C freezer
  8. Ice buckets
  9. 37 °C water bath
  10. Aspiration flasks (Safety-approved for radiolabeled materials)
  11. Head over head rotator
  12. A gel drying equipment

Software

  1. ImageJ

Procedure

  1. Harvest around 1 x 106 adherent cells by trypsinization or collect 1-5 ml of cells that grow in suspension. Cells should be in subconfluent conditions.
  2. Centrifuge cells for 5 min at 300 x g at room temperature and aspirate the medium.
  3. Resuspend cell pellet in DPBS and wash twice with 10 ml pre-warmed DPBS by centrifugation.
  4. Resuspend cell pellet in 200-500 μl of the pulse medium (see Recipes).
  5. Incubate cells for the desired duration of the pulse in a 37 °C water bath.
  6. Terminate the pulse by centrifugation at 4 °C for 15 sec at maximal speed.
  7. Aspirate the supernatant and resuspend the cell pellet in 1 ml of chase medium (37 °C, see Recipes).
  8. Take aliquots at several time points (4-8 time points are usually sufficient). Intervals between time points should be determined according to the estimated half-life of the protein of interest. Transfer the aliquots to new 1.5 ml micro-centrifuge tubes containing 1 ml of ice-cold DPBS and keep on ice.
  9. Centrifuge cells for 15 sec at maximal speed at 4 °C.
  10. Aspirate the supernatant and resuspend the cell pellet in appropriate volume of ice-cold lysis buffer (see Recipes) freshly supplemented with 1 mM DTT, protease inhibitor cocktail, phosphatase inhibitor cocktail, 1 mM N-ethylmaleimide (NEM). Usually 1 million cells per 100 μl of lysis buffer gives a suitable concentration of cell lysates.
  11. Incubate cell lysates for 20 min at 4 °C (cold room) under slow rotation (2-5 x g).
  12. Centrifuge lysate for 15 min at 13,000 x g at 4 °C to remove insoluble materials and cell debris.
  13. Transfer the clear lysates to a fresh tube and discard the pellet.
    Note: For adherent cells, this protocol can also be applied while cells are attached to the tissue culture dishes without trypsinization by using several dishes of cells at the same confluency, one for each time point. However, in that case the addition of pulse and chase media should be done quickly to avoid variation in pulse/chase time among dishes especially when applying short time intervals. Cell lysis in this case is done by adding the lysis buffer directly to cell monolayer while incubating on ice for 10 min then collecting the cell lysates using a scraper. Tubes containing the lysates are kept for another 10 min on ice with occasional inversion. Lysates are then centrifuged for 15 min at 13,000 x g at 4 °C to remove insoluble material and clear lysates are transferred to fresh tubes.
  14. Quantify protein concentration in total cell lysates from different aliquots/time points using Bradford colorimetric assay and adjust the concentration of lysates using freshly added lysis buffer to approximately 1 mg/ml.
  15. Continue with the immunoprecipitation step: Add antibody against the protein of interest at the recommended dilution for immunoprecipitation application together with 50 μl 10% Protein A-Sepharose beads and 0.5 mg of protein (in about 500 μl of lysate). Shake or rotate head over head. Incubate under constant rotation at 4 °C for 1-2 h or overnight at 4 °C (cold room) under slow rotation (2-5 x g).
  16. Pellet the immunoprecipitated complexes by centrifugation at 300 x g for 2 min at 4 °C.
  17. Remove the supernatant and wash the bead complexes 3-4 times by centrifugation at 300 x g for 2 min and discarding the supernatant and adding 1 ml ice cold wash buffer (see Recipes).
  18. Resuspend beads in 20 µl of 4x SDS sample buffer and boil samples on a 95 °C heat block for 5 min.
  19. Load samples onto SDS-PAGE gels.
    Note: The acrylamide percentage of acrylamide in the separation gel should be determined according to the size of protein of interest: the higher the molecular weight of the protein the less percentage gel to be used.
  20. Run at constant 50 mA for stacking gel and 70 mA for separation gel.
  21. Fix gels with 10 ml fixation solution (50% methanol + 10% acetic acid) for 1 h at room temperature with slow rocking.
  22. Dry gels in a gel dryer for 1.5 h at -80 °C.
  23. Expose films to the radioactive gels overnight at room temperature.
  24. Quantify images of films using imaging software such as ImageJ.

Data analysis

Pulse-chase method is a powerful tool to study protein folding, maturation, intracellular transport and degradation. A practical example to demonstrate the power and applicability of pulse-chase technique was recently shown in Elgendy et al., 2017. In this study, pulse-chase technique was used to study the modulation of the stability of MCL-1 oncoprotein upon treatment of cancer cells with Sunitinib. Sunitinib, a multikinase inhibitor, is one of the most widely used targeted therapy (Elgendy, 2017). Nevertheless, most patients eventually relapse secondary to sunitinib resistance (Cella et al., 2015). Our work demonstrated that sunitinib resistance is associated with enhanced stability of MCL-1 oncoprotein (Elgendy et al., 2017; Elgendy, 2017). MCL-1 is a pro-survival member of the Bcl-2 family of proteins that exhibits unique features including its tight regulations and short half-life (Elgendy et al., 2014; Elgendy and Minucci, 2015). Using pulse-chase method, sunitinib treatment has been shown to enhance the stability of MCL-1. Briefly, HCT116 cells were incubated in methionine/cysteine-free DMEM for 30 min followed by incubation in labeling medium containing 200-500 µCi of [35S] cysteine/methionine for 2 h at 37 °C. After labeling, the cells were chased with complete DMEM containing 10% FBS and 5 mM cold methionine at 37 °C and were either left untreated or were treated with sunitinib for the indicated time points. MCL-1 was then immunoprecipitated from the lysates and analyzed by autoradiography.

Notes

Immunoprecipitation protocol should be optimized for each protein of interest by fine tuning several variables to find the optimal combinations that give best signal to background ratio. The most important variables are:

  1. The composition of lysis and wash buffers, mainly the type and concentration of detergent used in the buffer as well as the stringency of the buffer. Some nuclear proteins and structural proteins will require harsh lysis conditions where the lysis buffer contains SDS.
  2. The concentrations of antibody used. Higher concentrations of the antibody can be used if the protein of interest is not so abundant.
  3. The amount of total lysates used. This is determined by the abundancy of the protein of interest in the cell type used. The higher the amount of lysates used the more non-specific binding to the beads and ultimately the higher the background will be.
  4. Including or excluding a pre-clearing step of the lysates by pre-incubation with beads alone or with control IgG without the specific antibody to get rid of the ‘sticky’ proteins that adhere non-specifically to the beads.
  5. The sequence and duration of incubation of antibody, beads and lysates. Usually shorter incubation time should be tried first to reduce the background which may be increased to overnight incubation at 4 °C if needed.
  6. The temperature and number of washing steps: the higher the temperature, the lower the background.

Recipes

  1. Pulse (labeling) medium
    Cysteine and methionine-free tissue-culture medium containing:
    10 mM HEPES, pH 7.4
    125-500 μCi [35S]-cysteine and/or methionine/ml
  2. Chase medium
    Complete tissue-culture medium containing:
    10 mM HEPES
    5 mM cysteine
    5 mM methionine
  3. Lysis buffer
    150 mM NaCl
    50 mM HEPES, pH 7.5
    1% Triton X-100
    1 mM EDTA
    10% glycerol
  4. Wash buffer
    20 mm Tris-HCl, pH 7.4
    150 mm NaCl
    0.1% SDS
    1 mm EDTA
  5. Fixation solution
    50% methanol
    10% acetic acid

Acknowledgments

This protocol was adapted from Elgendy et al. (2017). [Elgendy, M., Abdel-Aziz, A. K., Renne, S. L., Bornaghi, V., Procopio, G., Colecchia, M., Kanesvaran, R., Toh, C. K., Bossi, D., Pallavicini, I., Perez-Gracia, J. L., Lozano, M. D., Giandomenico, V., Mercurio, C., Lanfrancone, L., Fazio, N., Nole, F., Teh, B. T., Renne, G. and Minucci, S. (2017). Dual modulation of MCL-1 and mTOR determines the response to sunitinib. J Clin Invest 127(1): 153-168.] M.E .received funding from the Mahlke-Obermann Stiftung and the European Union's Seventh Framework Programme/FP7 Marie Curie Actions Grant Agreement No. 609431/INDICAR–Interdisciplinary Cancer Research.

References

  1. Cella, C. A., Minucci, S., Spada, F., Galdy, S., Elgendy, M., Ravenda, P. S., Zampino, M. G., Murgioni, S. and Fazio, N. (2015). Dual inhibition of mTOR pathway and VEGF signalling in neuroendocrine neoplasms: from bench to bedside. Cancer Treat Rev 41(9): 754-760.
  2. Elgendy, M. (2017). The yin yang of sunitinib: One drug, two doses, and multiple outcomes. Mol Cell Oncol 4(2): e1285385.
  3. Elgendy, M., Abdel-Aziz, A. K., Renne, S. L., Bornaghi, V., Procopio, G., Colecchia, M., Kanesvaran, R., Toh, C. K., Bossi, D., Pallavicini, I., Perez-Gracia, J. L., Lozano, M. D., Giandomenico, V., Mercurio, C., Lanfrancone, L., Fazio, N., Nole, F., Teh, B. T., Renne, G. and Minucci, S. (2017). Dual modulation of MCL-1 and mTOR determines the response to sunitinib. J Clin Invest 127(1): 153-168.
  4. Elgendy, M., Ciro, M., Abdel-Aziz, A. K., Belmonte, G., Dal Zuffo, R., Mercurio, C., Miracco, C., Lanfrancone, L., Foiani, M. and Minucci, S. (2014). Beclin 1 restrains tumorigenesis through Mcl-1 destabilization in an autophagy-independent reciprocal manner. Nat Commun 5: 5637.
  5. Elgendy, M. and Minucci, S. (2015). A novel autophagy-independent, oncosuppressive function of BECN1: Degradation of MCL1. Autophagy 11(3): 581-582.
  6. Jansens, A. and Braakman, I. (2003). Pulse-chase labeling techniques for the analysis of protein maturation and degradation. Methods Mol Biol 232: 133-145.
  7. Magadán, J. G. (2014). Radioactive pulse-chase analysis and immunoprecipitation. Bio Protoc 4(8).

简介

脉冲追踪技术是广泛用于评估蛋白质或mRNA稳定性的方法。 脉冲追踪的原理依赖于在短时间内产生的称为“脉冲”的蛋白质或mRNA的标记,然后遵循称为“追逐”的时间段内那些标记蛋白质的消失速率。 因此,该技术允许在不同处理或培养条件下对蛋白质或mRNA稳定性的调节进行定量分析。
【背景】脉冲追踪技术是一种方法,其涉及在称为“脉冲”的短时间内在含有标记氨基酸的培养基中培养细胞。这导致结合标记的氨基酸的新合成的多肽的产生。脉冲步骤之后是“追逐”步骤,其中洗涤标记培养基以终止标记过程,并被具有非标记氨基酸的培养基代替,以允许在任何给定时间定量初始合成的放射性标记的蛋白质在这个追逐阶段。因此,该技术可以及时地从合成到降解的方法定量分析感兴趣的蛋白质的加工。脉冲追踪方法可用于分析蛋白质折叠,共翻译修饰和细胞内运输等各种过程(Jansens和Braakman 2003;Magadán,2014)。然而,脉冲追踪技术最常用于评估不同实验条件下蛋白质的稳定性。
   放射性是常用的标签。标签通常用于使用放射性S35甲硫氨酸的蛋白质。放射性标记的蛋白质的消失动力学依赖于这些蛋白质降解的速度,可以用来检验不同实验条件对蛋白质稳定性的影响。为了评估特定目的蛋白质的稳定性,该蛋白质在使用特异性抗体在脉冲期间产生的所有其它免疫标记的蛋白质免疫沉淀。免疫沉淀将分离在脉冲期间产生并且直到免疫沉淀时尚未降解的放射性标记的蛋白质的比例。然后测量放射性以评估标记蛋白质在一段时间内的相对量。
   虽然该方案的重点是蛋白质稳定性的评估,但脉冲追踪方法也可用于评估mRNA的稳定性。脉冲步骤中mRNA的标记可以使用3UTP进行。然后通过在不同时间点获取总mRNA的等分试样并进行以下两种方法之一的定量分析:1)斑点印迹法,其中所标记的mRNA与附着在滤纸上的互补单链核酸的DNA退火然后洗涤其他标记的mRNA,并通过放射自显影或闪烁计数测量放射性; 2)亲和纯化方法,其中所关注的标记的mRNA与固定在珠上的互补锚定RNA / DNA的DNA退火,然后通过离心收集珠粒,捕获的标记的mRNA的放射性被量化。

关键字:脉冲追踪, 蛋白质稳定性, 半衰期, 蛋白质降解, 蛋白酶体, 溶酶体, MCL-1, mTOR, 舒尼替尼

材料和试剂

除了常规用于组织培养中的细胞和免疫沉淀和SDS-PAGE的材料之外,脉冲追踪实验所需的具体试剂是:

  1. 移液器提示
  2. 1.5 ml微量离心管
  3. 细胞培养皿
  4. 塑料电池刮板
  5. 组织培养基,适合所用的细胞系,不含甲硫氨酸和半胱氨酸
  6. DPBS
  7. 二硫苏糖醇(DTT)
  8. 蛋白酶抑制剂鸡尾酒
  9. 磷酸酶抑制剂混合物(Sigma-Aldrich,目录号:P2850)
  10. N-乙基马来酰亚胺(NEM)(Sigma-Aldrich,目录号:R3876)
    注意:本产品已停产。
  11. 布拉德福比色法测定
  12. 蛋白A-Sepharose珠粒
  13. [35 S] - 甲硫氨酸(PerkinElmer,目录号:NEG709A500UC)
  14. 甲硫氨酸(250mM的H 2 O,储存在-20℃)(Sigma-Aldrich,目录号:M5308)
  15. 半胱氨酸(500mM的H 2 O,储存在-20℃)(Sigma-Aldrich,目录号:C7352)
  16. 氯化钠(NaCl)
  17. Triton X-100
  18. 乙二胺四乙酸(EDTA)
  19. 甘油
  20. Tris-HCl
  21. 十二烷基硫酸钠(SDS)
  22. 甲醇
  23. 乙酸
  24. 脉冲(标签)介质(见配方)
  25. 追逐媒介(见食谱)
  26. 裂解缓冲液(见配方)
  27. 洗涤缓冲液(见配方)
  28. 固定溶液(见配方)

设备

  1. 移液器
  2. 冷藏离心机
  3. 95°C热块
  4. 细胞培养箱(37℃加湿5%CO 2)
  5. SDS-PAGE装置
  6. 射线胶片盒
  7. -80°C冰箱
  8. 冰桶
  9. 37°C水浴
  10. 吸入瓶(安全认可的放射性标记材料)
  11. 头顶旋转器
  12. 凝胶干燥设备

软件

  1. ImageJ的

程序

  1. 通过胰蛋白酶消化收获约1×10 6个贴壁细胞,或收集1-5ml悬浮液中生长的细胞。细胞应处于亚融合状态。
  2. 离心细胞在室温下以300×g离心5分钟并吸出培养基。
  3. 将细胞沉淀重悬于DPBS中,并通过离心用10ml预热的DPBS洗涤两次
  4. 重悬细胞沉淀在200-500μl脉冲培养基中(见食谱)。
  5. 在37°C水浴中孵育细胞所需的脉搏持续时间。
  6. 通过在4℃下以最大速度离心15秒终止脉冲。
  7. 吸出上清液并将细胞沉淀重悬于1ml追踪培养基(37℃,参见食谱)。
  8. 在几个时间点(4-8个时间点通常就足够了)。时间点之间的间隔应根据感兴趣的蛋白质的估计半衰期来确定。将等分试样转移到含有1ml冰冷DPBS的新的1.5ml微量离心管中,并保持在冰上。
  9. 离心细胞15秒,最高速度在4°C
  10. 吸出上清液并将新鲜补充有1mM DTT,蛋白酶抑制剂混合物,磷酸酶抑制剂混合物,1mM N-乙基马来酰亚胺(NEM)的适量体积的冰冷裂解缓冲液(参见Recipes)重悬悬浮细胞沉淀。每100μl裂解缓冲液通常有100万个细胞提供合适浓度的细胞裂解物
  11. 在缓慢旋转(2-5xg)下,在4℃(冷室)下孵育细胞裂解物20分钟。
  12. 在4℃以13,000 x g离心裂解物15分钟,以除去不溶物质和细胞碎片。
  13. 将澄清的裂解物转移到新鲜管中,并丢弃沉淀。
    注意:对于贴壁细胞,当将细胞连接到组织培养皿而不进行胰蛋白酶消化时,也可以应用该方案,通过在相同的汇合处使用几个细胞,每个时间点一个。然而,在这种情况下,应该快速添加脉冲和追踪介质,以避免菜肴中脉冲/追逐时间的变化,特别是在施加短时间间隔时。在这种情况下,细胞裂解是通过将裂解缓冲液直接加入到细胞单层中,同时在冰上孵育10分钟,然后使用刮刀收集细胞裂解物。将含有裂解物的管在冰上保持另外10分钟,偶尔倒置。然后将裂解物在4℃以13,000×g离心15分钟以除去不溶物质,将澄清的裂解物转移至新鲜管中。
  14. 使用Bradford比色法测定不同等分试样/时间点的总细胞裂解物中的蛋白质浓度,并使用新添加的裂解缓冲液将裂解物的浓度调节至约1mg / ml。
  15. 继续进行免疫沉淀步骤:将免疫沉淀应用的推荐稀释液与50μl10%蛋白A-Sepharose珠和0.5mg蛋白质(约500μl裂解物)一起添加到目标蛋白质上。摇头或旋转头顶。在4℃下恒温旋转1-2小时或在4℃(冷室)下缓慢旋转(2-5xg)过夜孵育。
  16. 通过在4℃下以300×g离心2分钟来将免疫沉淀的复合物颗粒化。
  17. 取出上清液,并以300×g离心3分钟洗涤珠子复合物2分钟,弃去上清液并加入1毫升冰冷的洗涤缓冲液(参见食谱)。
  18. 将珠重悬在20μl4x SDS样品缓冲液中,并在95°C热块上煮沸样品5分钟。
  19. 将样品加载到SDS-PAGE凝胶上。
    注意:分离凝胶中丙烯酰胺的丙烯酰胺百分比应根据感兴趣的蛋白质的大小来确定:蛋白质的分子量越高,凝胶的使用百分比越小。
  20. 以恒定的50mA运行堆叠凝胶,70mA用于分离凝胶。
  21. 用10ml固定溶液(50%甲醇+ 10%乙酸)固定凝胶1小时,缓慢摇摆。
  22. 干燥凝胶在凝胶干燥器中在-80℃下1.5小时。
  23. 在室温下将胶片暴露于放射性凝胶过夜。
  24. 使用ImageJ等成像软件量化电影图像。

数据分析

脉冲追踪法是研究蛋白质折叠,成熟,细胞内运输和降解的有力工具。最近在Elgendy等人,2017中展示了一种演示脉冲跟踪技术的功能和适用性的实例。在这项研究中,采用脉冲追踪技术来研究脉冲追踪技术的稳定性用舒尼替尼治疗癌细胞时MCL-1癌蛋白。舒尼替尼,多激蛋白抑制剂,是最广泛使用的靶向治疗之一(Elgendy,2017)。然而,大多数患者最终复发继发于舒尼替尼耐药(Cella et al。,2015)。我们的工作表明,舒尼替尼耐药性与MCL-1癌蛋白(Elgendy等人,2017; Elgendy,2017)的稳定性增强有关。 MCL-1是Bcl-2蛋白家族的主要生存成员,其表现出独特的特征,包括其严格的法规和短的半衰期(Elgendy等人,2014; Elgendy和Minucci,2015) )。采用脉冲追踪方法,舒尼替尼治疗已显示出增强MCL-1的稳定性。简言之,将HCT116细胞在含有甲硫氨酸/半胱氨酸的DMEM中孵育30分钟,然后在含有200-500μCi的[35℃]半胱氨酸/甲硫氨酸的标记培养基中于37℃孵育2小时。标记后,在37℃下用含有10%FBS和5mM冷甲硫氨酸的完全DMEM追踪细胞,并且在未指定的时间点内未处理或用舒尼替尼处理。然后将MCL-1从裂解物中免疫沉淀并通过放射自显影进行分析。

笔记

应对每种感兴趣的蛋白质优化免疫沉淀方案,通过微调几个变量,找到最佳信号与背景比的最佳组合。最重要的变量是:

  1. 裂解和洗涤缓冲液的组成,主要是缓冲液中使用的洗涤剂的类型和浓度以及缓冲液的严格性。一些核蛋白和结构蛋白将需要苛刻的裂解条件,其中裂解缓冲液含有SDS
  2. 所用抗体的浓度。如果感兴趣的蛋白质不那么丰富,可以使用更高浓度的抗体。
  3. 使用的总裂解物的量。这是由所使用的细胞类型中所关注的蛋白质的丰度确定的。使用的裂解物的量越高,对珠子的非特异性结合越多,背景越高。
  4. 通过与单独的珠子或与不含特异性抗体的对照IgG预孵育来包括或排除裂解物的预清除步骤,以除去非特异性粘附到珠粒上的"粘性"蛋白质。
  5. 抗体,珠粒和裂解液孵育的顺序和持续时间。通常应先尝试较短的孵化时间,以减少背景,如果需要,可以将其升高至4℃过夜孵育。
  6. 洗涤步骤的温度和数量:温度越高,背景越低

食谱

  1. 脉冲(标签)介质
    含有半胱氨酸和不含蛋氨酸的组织培养基含有:
    10 mM HEPES,pH 7.4
    125-500μCi[35] S半胱氨酸和/或甲硫氨酸/ ml
  2. 追逐媒介
    完整的组织培养基包含:
    10 mM HEPES
    5 mM半胱氨酸
    5 mM甲硫氨酸
  3. 裂解缓冲液
    150 mM NaCl
    50 mM HEPES,pH 7.5
    1%Triton X-100
    1 mM EDTA
    10%甘油
  4. 洗涤缓冲液
    20毫升Tris-HCl,pH 7.4 150毫米NaCl
    0.1%SDS
    1毫升EDTA
  5. 固定解决方案
    50%甲醇
    10%乙酸

致谢

该协议由Elgendy等人(2017)进行了改编。 [Elgendy,M.,Abdel-Aziz,AK,Renne,SL,Bornaghi,V.,Procopio,G.,Colecchia,M.,Kanesvaran,R.,Toh,CK,Bossi,D.,Pallavicini, Perez-Gracia,JL,Lozano,MD,Giandomenico,V.,Mercurio,C.,Lanfrancone,L.,Fazio,N.,Nole,F.,Teh,BT,Renne,G。和Minucci,S。(2017 )。 MCL-1和mTOR的双重调制决定了对于舒尼替尼的反应。 J Clin Invest 127(1):153-168。]我接受了Mahlke-Obermann基金会和欧盟第七框架计划/ FP7 Marie Curie行动赠款协议号609431 / INDICAR-跨学科癌症研究。

参考

  1. Cella,CA,Minucci,S.,Spada,F.,Galdy,S.,Elgendy,M.,Ravenda,PS,Zampino,MG,Murgioni,S。和Fazio,N。(2015)。 ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26142874"target ="_ blank">神经内分泌肿瘤中mTOR通路和VEGF信号传导的双重抑制:从长凳到床边。 癌症治疗 41(9):754-760。
  2. Elgendy,M.(2017)。  阴阳舒尼替尼:一种药物,两种剂量和多种结果。 Mol Cell Oncol 4(2):e1285385。
  3. Elgendy,M.,Abdel-Aziz,AK,Renne,SL,Bornaghi,V.,Procopio,G.,Colecchia,M.,Kanesvaran,R.,Toh,CK,Bossi,D.,Pallavicini,I.,Perez -Gracia,JL,Lozano,MD,Giandomenico,V.,Mercurio,C.,Lanfrancone,L.,Fazio,N.,Nole,F.,Teh,BT,Renne,G.and Minucci,S。(2017) 。 MCL-1和mTOR的双重调制决定了响应至舒尼替尼。 J Clin Invest 127(1):153-168。
  4. Elgendy,M.,Ciro,M.,Abdel-Aziz,AK,Belmonte,G.,Dal Zuffo,R.,Mercurio,C.,Miracco,C.,Lanfrancone,L.,Foiani,M.and Minucci,S 。(2014)。 Beclin 1通过Mcl- 1以自噬不相干的方式不稳定。 5:5637.
  5. Elgendy,M.和Minucci,S。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25837021"target ="_ blank" > BECN1的一种新颖的自噬不依赖的抑癌功能:MCL1的降解。 自噬 11(3):581-582。
  6. Jansens,A.和Braakman,I.(2003)。  用于分析蛋白质成熟和降解的脉冲追踪标记技术。方法Mol Biol 232:133-145。
  7. Magadán,JG(2014)。放射性脉冲追踪分析和免疫沉淀。 Bio Protoc 4(8)。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Elgendy, M. (2017). Assessment of Modulation of Protein Stability Using Pulse-chase Method. Bio-protocol 7(16): e2443. DOI: 10.21769/BioProtoc.2443.
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