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[Bio101] GST-Pull Down Protocol
[Bio101] GST-Pull Down 实验   

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Abstract

GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure.

Materials and Reagents

  1. pGEX plasmid (GE Healthcare Dharmacon)
  2. IPTG
  3. PBS
  4. Isoproponal
  5. Ethanol
  6. TritonTM X-100 
  7. SDS-PAGE
  8. Tryptone
  9. NaCl
  10. NaOH
  11. Agar
  12. Tris-HCl
  13. PMSF
  14. NaVO3
  15. NP-40 w
  16. Sodium-deoxycholate
  17. Commassive blue staining (Ezblue gel staining reagent) (Sigma-Aldrich, catalog number: G1041 )
  18. lysozyme (Sigma-Aldrich, catalog number: L7651 )
  19. Glutathione sepharose 4B (GE Healthcare Dharmacon, catalog number: 17-0756-01 )
  20. Protease inhibitors (Sigma-Aldrich, catalog number: P2741 )
  21. Phosphatase inhibitors (Sigma-Aldrich, catalog number: P2850 / P5726 )
  22. 2x YTA (see Recipes)
  23. Elution buffer (see Recipes)
  24. Lysis buffer (see Recipes)

Equipment

  1. Standard tabletop centrifuges
  2. Shaker
  3. Ultra Centrifugal Filter (EMD Millipore, catalog number: UFC900308 )
  4. Water bath
  5. Sonicator

Procedure

  1. Small-scale culture (pilot experiment to check if GST tagged protein is expressed properly)
    1. Growth and induction of cells
      1. Inoculate several colonies* containing pGEX-unp-ECD (unplugged extracellular domain expressing vector) into 2 ml 2x YTA medium (with amp).
      2. For comparison, inoculate a control tube with bacteria transformed with the parental pGEX plasmid.
      3. Grow the inoculum for 12-15 h at 37 °C with vigorous shaking. 
      4. Then dilute the culture 1:100 into 2 ml of 2x YTA medium (with amp).
      5. Grow liquid cultures to an A600 of 0.6-0.8 (about 3-5 h) with vigorous agitation at 20-37 °C.
      6. Induce fusion protein expression by adding 1-10 μl of 100 mM IPTG for each ml of culture volume (final concentration=0.1-1.0 mM).
      7. Continue incubation for an additional 1-2 h.
      8. Transfer the liquid cultures to Eppendorf tubes.
      9. Centrifuge to pellet cells (5 sec in a microcentrifuge) and discard the supernatants. Drain the pellets thoroughly and place tubes on ice.
      10. Resuspend each pellet in 50 μl of ice-cold 1x PBS for each ml of culture that centrifuged.
      11. Remove 10 μl of these resuspended cells into labeled tubes (for later use in SDS-PAGE analysis).
    2. Cell lysis (freeze/thaw)
      1. Prepare a 10 mg/ml lysozyme solution in water. Add 1 μl of lysozyme solution to each 100 μl of cell suspension.
      2. Vortex tubes gently to disperse lysozyme. Allow tubes to incubate at RT for 5 min.
      3. In a fume hood, prepare a dry ice bath in an ice bucket by adding dry ice and isoproponal until a slushy consistency is achieved. Prepare warm water bath in a separate ice bucket.
      4. Place tubes containing lysozyme-treated cell suspensions in the dry ice bath until cells are frozen solid for about 20 sec. 
      5. Transfer tubes to a flotation carrier and place in the warm water bath until the suspension becomes fully liquid, about 1 min. Repeat freeze/thaw cycle 10 times.
      6. Spin at full speed in a microcentrifuge for 10 min to remove insoluble material. Decant the supernatants into fresh tubes**. Save 10 μl aliquot of the insoluble and soluble material for analysis by SDS-PAGE.
    3. Preparation of glutathione sepharose 4B
      1. Gently shake the bottle of sepharose 4B to resuspend the matrix.
      2. Use a pipet to remove sufficient slurry for use and transfer to 15 ml falcon tube. (Dispense 1.33 ml of original sepharose slurry per ml of bed volume required.)
      3. Sendiment the matrix by centrifugation at 500 x g for 5 min. Carefully decant the supernatant.
      4. Wash the sepharose 4B by adding 10 ml of cold 1x PBS per 1.33 ml of the original slurry of glutathione sepharose dispensed. Invert to mix.
        Note: Sepharose 4B must be thoroughly washed with PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures.
      5. Sediment the matrix by centrifugation at 500 x g for 5 min. Decant the supernatant.
      6. For each 1.33 ml of the original slurry, add 1 ml of 1x PBS. This produces a 50% slurry. Mix well prior to the subsequent pipetting steps (Sepharose 4B equilibrated with PBS may be stored at 4 °C for up to a month).
    4. Purification of fusion proteins
      1. Add 20 μl of 50% slurry of glutathione sepharose 4B to each lysate supernatant and mix gently for 5 min at RT.
      2. Add 100 μl of 1x PBS, vortex briefly, and centrifuge fro 5 sec to sediment the sepharose beads.
      3. Discard the supernatants. Repeat this 1x PBS wash twice for a total of 3 washes.
      4. Elute the fusion protein by the addition of 10 μl of glutathione elution buffer. Suspend the sepharose beads and incubate at RT for 5 min.
      5. Centrifuge for 5 min to sendiment the sepharose beads, and transfer the supernatants to fresh tubes.
    5. Analysis of fusion proteins
      1. Use 10 μl aliquots of each sample to run the SDS-gel.
      2. Stain with Coomassie blue to visualize the parental pGEX and the fusion protein. GST band is 29 kDa. 
      3. Or, perform western blots using anti-GST antibody.
        *The bacteria can be grown on LB+Amp plates, but better be grown on 2x YTA+Amp plates.
        **If the lysate is too viscous for handling. Add DNase I to a final concentration of 10 μg /ml in step 9.

  2. Large-scale culture (for GST binding assay)
    1. Growth and induction of cells
      1. Inoculate a single colony containing a recombinant pGEX-unp-ECD or pGEX into 2 ml 2x YTA medium (with amp).
      2. Grow the inoculum for 12-15 h at 37 °C with vigorous shaking. 
      3. Then dilute the culture 1:100 into desired volume of 2x YTA medium (with amp). (I usually grow 400 ml culture, so I combine two small cultures).
      4. Grow liquid cultures to an A600 of 0.6-0.8 (about 2.5-3 h) with vigorous agitation at 37 °C.
      5. Induce fusion protein expression by adding the desired volume of 100 mM IPTG to the culture (final concentration=1.0 mM).
      6. Continue incubation for an additional 1 h at low temperature (30 °C).
      7. Transfer the liquid cultures to labeled centrifuge tubes. 
      8. Centrifuge to pellet cells (7,700 x g for 10 min) and discard the supernatants. Drain the pellets thoroughly and place tubes on ice.
      9. Resuspend each pellet in 50 μl of ice-cold 1x PBS for each ml of culture that centrifuged (20 ml iced 1x PBS for 400 ml cultures). 
      10. Remove 10 μl of these resuspended cells into labeled tubes (for later use in SDS-PAGE analysis).
    2. Cell lysis (sonication)
      1. Lyse the cells using a sonicator equipped with an appropriate probe.
        Note: I usually do lysozyme treatment before sonication (1 μl of lysozyme, 10 mg/ ml in H2O, to 100 μl of cell suspension), freeze and thaw using dry ice and warm water bath for 3 times. 
      2. Sonicate 10-20 sec each time and totally 10-15 times.
      3. Lysis is complete when the cloudy cell suspension becomes translucent. The frequency and intensity of sonication should be adjusted such that complete lysis occurs in 10 sec without frothing (which can denature proteins). The extent of lysis maybe monitored by microscopic examination of culture sonicates.
      4. Large-scale sonicates may require the addition of 20% TritonTM X-100 to a final. 
      5. Centrifuge at 8,000 rpm for 10 min to remove insoluble material. Transfer the supernatants to fresh tubes. Save a 10 μl aliquot of the insoluable material for SDS-PAGE.
    3. Preparation of glutathione sepharose 4B
      1. Gently shake the bottle of sepharose 4B to resuspend the matrix.
      2. Use a pipet to remove sufficient slurry for use and transfer to an 15 ml falcon tube (dispense 1.33 ml of original sepharose slurry per ml of bed volume required).
      3. Sendiment the matrix by centrifugation at 500 x g for 5 min. Carefully decant the supernatant.
      4. Wash the sepharose 4B by adding 10 ml of cold 1x PBS per 1.33 ml of the original slurry of glutathione sepharose dispensed. Invert to mix. (Sepharose 4B must be thoroughly washed with PBS to remove the 20% ethanol storage solution. Residual ethanol may interfere with subsequent procedures).
      5. Sediment the matrix by centrifugation at 500 x g for 5 min. Decant the supernatant. 
      6. For each 1.33 ml of the original slurry, add 1 ml of 1x PBS. This produces a 50% slurry. Mix well prior to the subsequent pipetting steps (sepharose 4b equilibrated with PBS may be stored at 4 °C for up to a month.
    4. Purification of fusion proteins
      1. Add 2 ml of a 50% slurry of glutathione sepharose 4B equilibrated with 1x PBS to each 100 ml of sonicates (400 μl of 50% slurray to 20 ml sonicates).
      2. Incubate with gentle agitation at RT for 1 h. 
      3. Centrifuge at 500 x g (1,400 rpm) for 5 min. Remove the supernatant and save a small volume for analysis by SDS-PAGE to measure the efficiency of binding to the matrix. 
      4. Wash the matrix with 10 bed volumes of 1x PBS (2 ml 1x PBS for 400 μl 50% slurray). Centrifuge at 500 x g for 5 min. Remove the supernatant. Repeat twice more for a total of three washes. 
      5. In the last wash or after 3 washes, the lysis buffer* in which the pull-down experiment will be performed can be used instead. 
      6. After last washing extensively, add the lysis buffer to the GST-fusion protein/sepharose 4B beads, resulting a 50% slurry. Keep the beads on ice. (can add 100 μg ml-1 BSA to reduce the nonspecific binding) (200 μl 1x PBS+PIs).
      7. Take 20 μl out for SDS-PAGE gel and the coomassie staining to measure the amount of bound protein. Dilute GST protein in different folds to get similar amounts as for GST fusion protein in order to find the right dilution.
    5. Preparation of Wnt conditional medium
      1. Grow 293T cells and transfect the cells with Wnt expressing construct and incubate for 3-5 days. Wnt protein will be secreted into the medium.
      2. Harvest medium and centrifuge at 1,000 x g (2,000 rpm) for 10 min to remove cells. 
      3. Filter through a nitrocellulose membrane (0.2 microcentimeter). 
      4. Concentrate the medium through millipore ultra centrifugal fliter (10 kDa).
        For a swinging rotor, spin at maximum 4,000 x g for 15-45 min.
        For 35 degree fixed angle rotor, spin at maximum 5,000 x g for 15-45 min.
        Leave the volume gradations facing up.
        For pre-rinse the centrifugal filter, add deionized water and spin for a couple of minutes, don’t leave the membrane dry.
      5. Take the concentrate out by using the thin-end tip. and add lysis buffer to 1 ml.
    6. Preclear the wnt conditioned medium
      1. Preclear the medium by adding about 15 μl of undiluted GST beads to 1 ml medium and incubate at 4 °C for 2 h.
        (For both control medium and wnt11rFLAG medium.)
    7. Pull-down experiments
      1. Add 20 μl of GST-SV1-ECD beads to 1 ml precleared medium.
        Incubate at 4 °C for 2 h to O/N. (totally 2-5 μg of fusion protein**).
        Dilute GST-beads with naked beads appropriately (to control the same amount of input for GST and GST fusion protein). And add the same amount of beads to the medium.
      2. Centrifuge at 500 x g for 5 min and remove the supernatants.
      3. Wash the beads using lysis buffer* for 3-4 times. e.g. 50 μl of beads need 1 ml lysis buffer per wash.
        200 μl lysis buffer for 20 μl beads. 
      4. Add 2x SDS loading buffer to the beads and then boil for 10 min. e.g. 50 μl of beads need 30-40 μl loading buffer.
    8. Analysis of fusion proteins
      1. Use 10 μl aliquots of each sample to run the SDS-gel. Stain with Coomassive blue to visualize the parental pGEX and the fusion protein. GST protein is 29 kDa.
        ** determine the amount of protein bound to the beads per volume.
        Take 20 μl of beads out (from step 20) and then eluted with loading buffer, boil and then run SDS-PAGE gel. As a maker, run BSA with different amounts alongside. Stain the gel with G-blue and then determine the amount of protein attached to the beads per 20 μl volume.

Recipes

  1. 2x YTA
    tryptone 16 g/L
    yeast extract 10 g/L
    NaCl 5 g/L
    Adjust pH to 7.0 with NaOH. Autoclave for 20 min. Add antibiotics when the medium has cooled. To prepare a solid mediu, add 12-15 g of agar prior to autoclaving.
  2. Elution buffer
    0.154 g reduced glutathione to 50 ml of dilution buffer (10 mM glutathione, pH 8.0).
    Shake gently until the powder is completely dissolved. Dispense into 1-10 ml aliquots, and store at -20 °C until needed. Avoid more than 5 freeze/thaw cycles.
    Note: Dilution buffer: 50 mM Tris-HCl (pH 8.0).
  3. IPTG solution
    100 mM in sterile water. Dispense as 1 ml aliquots and store at -20 °C.
  4. *Lysis buffer
    1x PBS (pH 7.4)
    1% Triton-X-100
    1x protease inhibitors
    1x Phosphatase inhibitors
    1 mM PMSF
    1 mM NaVO3
  5. Or RIPA buffer can be used instead of lysis buffer
    50 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1% NP-40
    0.25% sodium-deoxycholate
    Proteinase inhibitors

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.

简介

GST-下拉测定法是检查两种蛋白质在体外的直接结合的有效方法。 该协议基于GE Healthcare的GST下拉系统,并使用未插入的/MuSK受体和Wnt配体的结合作为例子来说明详细的过程。

材料和试剂

  1. pGEX质粒(GE Healthcare Dharmacon)
  2. IPTG
  3. PBS
  4. 异丙酚
  5. 乙醇
  6. Triton TM X-100 
  7. SDS-PAGE
  8. 胰蛋白酶
  9. NaCl
  10. NaOH
  11. 琼脂
  12. Tris-HCl
  13. PMSF
  14. NaVO <3>
  15. NP-40w
  16. 脱氧胆酸钠
  17. 标记蓝染色(Ezblue凝胶染色试剂)(Sigma-Aldrich,目录号:G1041)
  18. 溶菌酶(Sigma-Aldrich,目录号:L7651)
  19. 谷胱甘肽琼脂糖4B(GE Healthcare Dharmacon,目录号:17-0756-01)
  20. 蛋白酶抑制剂(Sigma-Aldrich,目录号:P2741)
  21. 磷酸酶抑制剂(Sigma-Aldrich,目录号:P2850/P5726)
  22. 2x YTA(见配方)
  23. 洗脱缓冲液(见配方)
  24. 裂解缓冲液(见配方)

设备

  1. 标准台式离心机
  2. 摇床
  3. 超离心过滤器(EMD Millipore,目录号:UFC900308)
  4. 水浴
  5. 超声波仪

程序

  1. 小规模培养(试验性实验来检查GST标记的蛋白质是否被正确表达)
    1. 细胞的生长和诱导
      1. 将含有pGEX-unp-ECD(未阻塞的胞外结构域表达载体)的几个菌落接种到2ml 2x YTA培养基(含有amp)中。
      2. 为了比较,用用亲本pGEX质粒转化的细菌接种对照管。
      3. 在37℃下剧烈振荡使接种物生长12-15小时。
      4. 然后将培养物1:100稀释到2ml的2x YTA培养基(含有amp)中。
      5. 在20-37℃下剧烈搅拌下将液体培养物生长至0.6-0.8(约3-5小时)的A 600。
      6. 通过每毫升培养体积(终浓度= 0.1-1.0mM)加入1-10μl100mM IPTG诱导融合蛋白表达。
      7. 继续孵育另外1-2小时。
      8. 将液体培养物转移到Eppendorf管中。
      9. 离心以沉淀细胞(在微量离心机中5秒)并弃去上清液。 彻底排空小丸,将管置于冰上。
      10. 重悬在每个沉淀在50μl冰冷的1×PBS每离心培养的毫升。
      11. 取出10微升的这些重悬细胞到标记的管(以后用于SDS-PAGE分析)
    2. 细胞裂解(冷冻/解冻)
      1. 准备10mg/ml溶菌酶的水溶液。 加入1微升的溶菌酶溶液到每100微升的细胞悬浮液。
      2. 涡旋管轻轻地分散溶菌酶。 允许管在室温孵育5分钟。
      3. 在通风橱中,通过加入干冰和异丙醇制备在冰桶中的干冰浴,直到达到泥浆稠度。 准备温水浴在一个单独的冰桶。
      4. 将含有溶菌酶处理的细胞悬浮液的管置于干冰浴中,直到细胞冷冻固体约20秒。
      5. 将管转移到浮选载体,并放置在温水浴中,直到悬浮液变成完全液体,约1分钟。 重复冷冻/解冻循环10次。
      6. 在微量离心机中全速旋转10分钟以除去不溶性物质。 滗析上清液到新鲜管**。 保存10微升的不溶性和可溶性材料的等分试样,通过SDS-PAGE分析
    3. 谷胱甘肽琼脂糖4B的制备
      1. 轻轻摇动瓶sepharose 4B重悬基质。
      2. 使用移液管移除足够的浆液使用,并转移到15毫升falcon管。 (需要1.33ml原始琼脂糖浆/ml床体积)。
      3. 通过在500×g离心5分钟来使基质再生。小心倾析上清液。
      4. 通过加入10ml冷的1x PBS每1.33ml的分配的谷胱甘肽琼脂糖的原始浆液洗涤sepharose 4B。反转混合。
        注意:Sepharose 4B必须用PBS彻底清洗以除去20%乙醇储存溶液。残留乙醇可能会干扰后续程序。
      5. 通过在500×g离心5分钟沉淀基质。滗析上清液。
      6. 对于每个1.33ml的原始浆液,加入1ml的1×PBS。这产生50%的浆料。在随后的移液步骤之前充分混合(用PBS平衡的Sepharose 4B可以在4℃下储存长达一个月)。
    4. 融合蛋白的纯化
      1. 向每个裂解液上清液中加入20μl50%的谷胱甘肽琼脂糖4B浆液,并在室温下轻轻混合5分钟。
      2. 加入100μl1×PBS,短暂涡旋,离心5秒,沉淀sepharose珠。
      3. 弃去上清液。 重复此1x PBS洗涤两次,共3次洗涤。
      4. 通过加入10μl的谷胱甘肽洗脱缓冲液洗脱融合蛋白。 悬浮琼脂糖珠,并在室温下孵育5分钟。
      5. 离心5分钟以移去sepharose珠,并将上清液转移到新管中
    5. 融合蛋白的分析
      1. 使用每个样品的10微升等分试样运行SDS凝胶。
      2. 用考马斯亮蓝染色以显示亲本pGEX和融合蛋白。 GST条带为29 kDa。
      3. 或者,使用抗GST抗体进行Western印迹 *细菌可以在LB + Amp平板上生长,但最好在2x YTA + Amp平板上生长。
        **如果裂解物太粘而不能处理。 在步骤9中加入DNase I至终浓度为10μg/ml
  2. 大规模培养(用于GST结合测定)
    1. 细胞的生长和诱导
      1. 将含有重组pGEX-unp-ECD或pGEX的单个菌落接种到2ml 2x YTA培养基(带有amp)中。
      2. 在37℃下剧烈振荡使接种物生长12-15小时。
      3. 然后将培养物1:100稀释到所需体积的2x YTA培养基(用amp)中。 (我通常生长400毫升文化,所以我结合两个小文化)。
      4. 在37℃剧烈搅拌下将液体培养物生长至0.6-0.8(约2.5-3小时)的A 600。
      5. 通过向培养物中加入所需体积的100mM IPTG诱导融合蛋白表达(终浓度= 1.0mM)。
      6. 继续在低温(30℃)下孵育另外1小时。
      7. 将液体培养物转移到标记的离心管中。
      8. 离心以沉淀细胞(7,700×g,10分钟),弃去上清液。 彻底排空小丸,将管置于冰上。
      9. 重悬在每个沉淀在50微升的冰冷的1×PBS的每毫升离心的文化(20毫升1×PBS的400毫升培养物)。
      10. 取出10微升的这些重悬细胞到标记的管(以后用于SDS-PAGE分析)
    2. 细胞裂解(超声处理)
      1. 使用配有适当的探头的超声波仪裂解细胞。
        注意:我通常在超声处理之前进行溶菌酶处理(1μl溶菌酶,在H 2 O中10mg/ml至100μl细胞悬浮液),使用干冰和温热进行冷冻和解冻 水浴3次。
      2. 每次超声10-20秒,共10-15次。
      3. 当混浊的细胞悬浮液变得半透明时,裂解完成。 应该调节超声处理的频率和强度,使得完全裂解在10秒内发生,不发泡(其可以使蛋白质变性)。 可以通过培养物声处理的显微镜检查来监测裂解的程度。
      4. 大规模声波处理可能需要添加20%的TritonTM X-100才能达到最终效果。
      5. 以8,000rpm离心10分钟以除去不溶性物质。 将上清液转移到新鲜管中。 在SDS-PAGE中保存10μl不溶性物质的等分试样
    3. 谷胱甘肽琼脂糖4B的制备
      1. 轻轻摇动瓶sepharose 4B重悬基质。
      2. 使用移液管移除足够的浆液使用,并转移到15毫升falcon管(分配1.33毫升原始sepharose浆液每ml床所需的体积)。
      3. 通过在500×g离心5分钟使基质再生。 小心倾析上清液。
      4. 通过加入10ml冷的1x PBS每1.33ml的分配的谷胱甘肽琼脂糖的原始浆液洗涤sepharose 4B。 反转混合。 (Sepharose 4B必须用PBS彻底洗涤 以去除20%乙醇储存溶液。 残留乙醇可能干扰后续程序)。
      5. 通过在500×g离心5分钟沉淀基质。 滗析上清液。
      6. 对于每个1.33ml的原始浆液,加入1ml的1×PBS。 这产生50%的浆料。 在随后的移液步骤之前充分混合(用PBS平衡的sepharose 4b可以在4℃下储存长达一个月。
    4. 融合蛋白的纯化
      1. 加入2ml用1×PBS平衡的谷胱甘肽琼脂糖凝胶4B的50%浆液到每100ml声处理液中(400μl50%浆液到20ml声处理)。
      2. 在室温下温和搅拌孵育1小时。
      3. 在500×g(1400rpm)离心5分钟。除去上清液并保存小体积用于通过SDS-PAGE分析以测量与基质结合的效率。
      4. 用10床体积的1×PBS(2ml 1×PBS,400μl50%浆液)洗涤基质。在500×g离心5分钟。除去上清液。重复两次,共三次洗涤。
      5. 在最后一次洗涤或3次洗涤后,可以使用进行下拉实验的裂解缓冲液*。
      6. 最后大量洗涤后,将裂解缓冲液加入GST-融合蛋白/琼脂糖4B珠中,得到50%浆液。保持小珠在冰上。 (可以加入100μg/ml BSA以减少非特异性结合)(200μl1×PBS + PI)。
      7. 取20μl用于SDS-PAGE凝胶和考马斯染色以测量结合蛋白的量。稀释GST蛋白在不同的折叠,以获得类似量的GST融合蛋白,以找到正确的稀释。
    5. Wnt条件培养基的准备
      1. 生长293T细胞,用Wnt表达构建体转染细胞,孵育3-5天。 Wnt蛋白将分泌到培养基中。
      2. 收获培养基并在1,000×g(2,000rpm)离心10分钟以除去细胞。
      3. 通过硝酸纤维素膜(0.2微米)过滤。
      4. 通过毫升超离心Fliter(10 kDa)浓缩培养基 对于摆动转子,最大旋转4,000 x g,持续15-45分钟。
        对于35度固定角转子,旋转最大5,000 x g,15-45分钟。
        使音量等级面朝上。
        对于预冲洗离心过滤器,加入去离子水并旋转几分钟,不要将膜干燥。
      5. 使用细末端提取浓缩液。 并加入裂解缓冲液至1ml
    6. 预清楚wnt条件培养基
      1. 通过向1ml培养基中加入约15μl未稀释的GST珠来预清洗培养基,并在4℃下孵育2小时。
        (对于对照培养基和wnt11rFLAG培养基。)
    7. 下拉实验
      1. 加入20微升GST-SV1-ECD珠到1毫升预清除的培养基。
        在4°C孵育2小时至O/N。 (总共2-5μg融合蛋白**) 用裸珠适当稀释GST-珠(以控制相同量的GST和GST融合蛋白的输入)。 并向培养基中加入相同量的珠子。
      2. 在500×g离心5分钟,除去上清液
      3. 使用裂解缓冲液*洗涤珠3-4次。 例如 50μl珠子需要每次洗涤1ml裂解缓冲液。
        200μl裂解缓冲液,用于20μl珠子。
      4. 向珠子中加入2x SDS上样缓冲液,然后煮沸10分钟。 例如 50微升的珠需要30-40微升的上样缓冲液
    8. 融合蛋白的分析
      1. 使用每个样品的10微升等分试样运行SDS凝胶。 用Coomassive blue染色以显示亲本pGEX和融合蛋白。 GST蛋白为29kDa **确定每单位体积与珠子结合的蛋白质量 取20μl的珠子(从步骤20),然后用上样缓冲液洗脱,煮沸,然后运行SDS-PAGE凝胶。 作为制造商,运行不同数量的BSA。 用G-蓝染色凝胶,然后测定每20μl体积连接到珠上的蛋白质的量

食谱

  1. 2x YTA
    胰蛋白胨16 g/L
    酵母提取物10g/L
    NaCl 5g/L
    用NaOH调节pH至7.0。 高压灭菌20分钟。 当介质冷却时加入抗生素。 为了制备固体培养基,在高压灭菌之前加入12-15g琼脂
  2. 洗脱缓冲液
    0.154g将谷胱甘肽还原成50ml稀释缓冲液(10mM谷胱甘肽,pH 8.0) 轻轻摇动直到粉末完全溶解。 分配到1-10毫升等分试样,并存储在-20°C直到需要。 避免超过5个冻/融循环。
    注意:稀释缓冲液:50 mM Tris-HCl(pH 8.0)。
  3. IPTG解决方案
    100mM在无菌水中。 分配为1ml等分试样并储存在-20℃
  4. *裂解缓冲液
    1x PBS(pH 7.4)
    1%Triton-X-100 1x蛋白酶抑制剂
    1×磷酸酶抑制剂
    1mM PMSF
    1mM NaVO 3
  5. 或者可以使用RIPA缓冲液代替裂解缓冲液
    50mM Tris-HCl(pH7.4) 150mM NaCl 1%NP-40
    0.25%脱氧胆酸钠 蛋白酶抑制剂

致谢

该协议是在美国宾夕法尼亚大学的Michael Granato实验室开发的,这项工作由NIH授权R01HD037975支持。

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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). GST-Pull Down Protocol. Bio-protocol Bio101: e177. DOI: 10.21769/BioProtoc.177;
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Jana M
tcd
Hi,
I have a silly question. When I pull down protein of my interest with my gst-fusion protein bound to glutathion sepharose 4b beads (for example TLR3-GST beads) what do I use as control beads? Is it glutathion sepharose 4b slurry (naked beads) or glutathion sepharose beads bound to GST protein (GST beads)?
thanks a lot for answer!
Jana
11/5/2013 8:05:18 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

It should be GST beads.

11/8/2013 5:58:08 PM


Reyhan Salehi
mcgill
Is the pGEX4T3 plasmid good for bait protein expression?
7/10/2013 11:51:04 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

YES.

7/10/2013 6:35:54 PM


Reyhan Salehi
mcgill

Thank you!

7/11/2013 10:47:50 AM


2/9/2013 3:42:04 PM Reply
Good afternoon,

How can I decrease background in GST-pulldown assay. I have been told I could use BSA.
At what stage of the protocol? How long, what concentration..?

In anticipation, thanks for your help,

Edith
(Brussels University)
1/28/2013 6:36:06 AM Reply
Jian-Guo Zhang
Cancer and Haematology Division and Department of Medical Biology, The Walter and Eliza Hall Institute of Medical Research and The University of Melbourne, Australia

Edith: Not sure how strong your specific signal was in your GST-pulldown assay? If it was strong, in my opinion, the best way to reduce the non-specific background is to reduce the amount of Glutathione-Sepharose beads you were using in your experiment? A colleague you've never met.

2/9/2013 4:04:45 PM


Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Sorry for the belated response.

To reduce the background, you can try to use smaller amount of beads, shorten the incubation time and prolong the washing time.

You can also use BSA to pre-block the beads. For example, you can use 1-5% BSA (in your suitable buffer) to incubate beads at 4 degrees for a couple of hours to O/N. Then wash in buffer a couple of times before the addition of the protein lysates.

Hope it helps.

2/28/2013 5:42:56 PM