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IEF-2DE Analysis and Protein Identification
IEF-2DE 分析和蛋白质鉴定   

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Abstract

Isoelectric focusing followed by SDS-PAGE (IEF-2DE) separates proteins in a two-dimensional matrix of protein pI (Protein Isoelectric Point) and molecular weight (MW). The technique is particularly useful to distinguish protein isoforms (Radwan et al., 2012) and proteins that contain post-translational modifications such as phosphorylation (Oh et al., 2012) and lysine acetylation (Wu et al., 2011). Proteins that are separated by IEF-2DE can be identified by immunoblot analysis using sequence-specific antibodies or by mass spectrometry.

Materials and Reagents

  1. Protein extracts
  2. Phenol (TE-saturated pH 7.9) (Sigma-Aldrich, catalog number: P4557 )
  3. 0.1 M ammonium acetate (dissolved in methanol)
  4. Tris-base (General Electric Company, catalog number: 17-1321-01 )
  5. Glycine
  6. Urea (Life Technologies, Invitrogen™, catalog number: ZU10001 )
  7. Thiourea (Life Technologies, Invitrogen™, catalog number: ZT10002 )
  8. CHAPS (Life Technologies, Invitrogen™, catalog number: ZC10003 )
  9. DTT (General Electric Company, catalog number: 17-1318-01 )
  10. Bromophenol blue (General Electric Company, catalog number: 17-1329-01 )
  11. Glycerol (General Electric Company, catalog number: 17-1325-01 )
  12. SDS (General Electric Company, catalog number: 17-1313-01 )
  13. Iodoacetamide (General Electric Company)
  14. 100% Ethanol (HPLC grade)
  15. Water (HPLC grade)
  16. IPG buffer (General Electric Company, catalog number: 17-6000-87 )
  17. IPG strips (General Electric Company, catalog number: 17-6000-14 )
  18. Immobiline DryStrip Cover Fluid (General Electric Company, catalog number: 17-1335-01 )
  19. Bradford assay dye reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
  20. SimplyBlueTM SafeStain protein stain (Life Technologies, Invitrogen™, catalog number: LC-6065 )
  21. Precision PlusTM All Blue Protein Standards (Bio-Rad Laboratories, catalog number: 161-0373 )
  22. IEF sample buffer (see Recipes)
  23. SDS equilibration buffer (see Recipes)
  24. 10x Tank buffer (see Recipes)
  25. Agarose overlay solution (General Electric Company, catalog number: 17-0554-01 ) (see Recipes)

Equipment

  1. 1.5 ml microfuge tubes
  2. 15 ml tubes
  3. EttanTM IPGphoreTM Isoelectric Focusing System (General Electric Company, Model: 80-6414-02 )
  4. Large Format 1-D Electrophoresis Systems (Bio-Rad Laboratories)
  5. Semi-dry transfer system (Bio-Rad Laboratories)
  6. Mass spectrometers (such as MALDI-TOF or tandem mass spectrometer)

Procedure

  1. Sample preparation
    To maximize the separation of proteins by IEF, the protein extracts should contain minimal contaminants of salts and detergents. Here is the method we used to prepare protein extracts (protein extraction buffer: 100 mM Tris-HCl, pH 8.0) for IEF analysis.
    1. Mix crude protein extract and ice-cold phenol (TE-saturated, pH 7.9) in a 1:1 (v:v) ratio. Vortex for 1 min.
    2. Centrifuge the mixture at 24,000 x g for 15 min at 4 °C.
    3. Carefully remove upper aqueous phase and discard (do not disturb interphase).
    4. Add equal volume of ice-cold 50 mM Tris-HCl (pH 8.0) to the protein mixture. Vortex for 1 min.
    5. Centrifuge at 24,000 x g for 15 min at 4 °C.
    6. Remove upper aqueous phase and discard (do not disturb interphase).
    7. Repeat steps 4 to 6 once more.
    8. Transfer the lower phenol phase (including interphase) to a new ice-cold 15 ml tube and add 5 volumes of ice-cold 0.1 M ammonium acetate (in methanol). Vortex for 1 min.
    9. Precipitate proteins overnight at -20 °C.
    10. Gently and briefly vortex to resuspend the protein precipitate. Split the samples between 1.5 ml microfuge tubes as necessary.
    11. Centrifuge at 24,000 x g for 20 min at 4 °C and carefully remove the supernatant.
    12. Add 1 ml ice-cold 0.1 M ammonium acetate (in methanol) to each microfuge tube.
    13. Let pellet “soak” in the buffer for 10 min at -20 °C.
    14. Centrifuge at 24,000 x g for 20 min at 4 °C.
    15. Pipette out the supernatant and take care to not disturb the pellet.
    16. Repeat steps 12 to 15 twice.
    17. Wash each pellet further with 1 ml ice-cold 100% ethanol.
    18. Centrifuge at 24,000 x g for 20 min at 4 °C.
    19. Pipette out the supernatant (as much as possible).
    20. Resuspend each protein pellet with the volume of about 50-100 μl IEF sample buffer. The total resuspension volume per sample is about 500 μl.
    21. Vortex the pellet and let it dissolve in IEF sample buffer at room temperature for 1 h. Do not heat the pellet in IEF sample buffer.
    22. Centrifuge at 24,000 x g for 20 min at room temperature. The resulting supernatant is suitable for IEF analysis.
    23. Quantify the protein concentration with the Bradford assay. Store protein extract at -80 °C until use.

  2. IEF Protein separation
    1. Thaw the protein extract at room temperature.
    2. Centrifuge the protein extract at 24,000 x g for 20 min at room temperature to remove any precipitate.
    3. Take an aliquot containing 200 μg of total protein and adjust the volume to 250 μl with IEF sample buffer.
      Note: A good range of sample loading for a 13 cm IPG strip is about 100 – 250 μg total proteins. Generally, we used 13 cm IPG strip (pH 3-10), but we can choose the strip depend on proteins analyzing.
    4. Load 250 μl of each sample uniformly into the ceramic strip holder (13 cm size).
    5. Take out a 13 cm IPG strip and remove the thin plastic seal from the gel side of the strip.
      Notes:
      1. The pH range of the strip should match the pH range of the IPG buffer in the IEF sample buffer.
      2. Use a tweezer to handle IPG strip. Avoid touching the gel area of the strip.
    6. Carefully place the IPG strip onto the surface of the IEF sample in the ceramic strip holder, with gel-side down and the acidic end of the strip (marked with a “+”) towards the pointed (+) end of strip holder (Figure 1A).


      Figure 1. Illustration of IEF-2DE protein separation by protein pI and MW. A. In the first dimension, proteins are separated by protein pI in IEF strip. B. IEF setup to show the positive side the IEF gel should match the arrow direction of ceramic strip holder and the IPGphor positive electrode plate. C. In the second dimension, proteins are further separated by protein MW in SDS-PAGE.

    7. Carefully raise and lower the strip to assure the gel side of IPG strip is in good contact with the IEF sample solution.
    8. Overlay strip with about 1.5 ml Immobiline DryStrip Cover Fluid.
    9. Cover the strip holder with the plastic lid.
    10. Place the strip unit onto the IPGphor Manifold with pointed (+) end on the large “+” electrode plate.
    11. Begin IEF protocol #1 (Voltage-assisted rehydration sample loading) 20 °C, 50 μA per strip, 0 h rehydration (under non-voltage).
      Note: Ensure that the correct number of IPG strips to be run is entered.
      Step 1 = 30 V for 360 voltage-hour (Vh)
      Step 2 = 500 V for 500 Vh
      Step 3 = 1,000 V for 1,000 Vh
      Step 4 = 8,000 V for 32,000 Vh
      Step 5, 10 h hold at 8,000 V
      Total running time = about 20 h (This range of voltage-hour recommended for all pH ranges of IPG strip)
    12. The IEF separation is completed. A good range to stop is at the 32,000 Vh to 42,000 Vh.
    13. Proceed directly to IPG strip equilibration (described below) or store the strip in a closed container before processing further at -80 °C.

  3. SDS-PAGE Protein separation
    1. Pour a 1 mm x 20 cm sized 10% acrylamide gel (pH 8.8 or 9.2) using a flat comb with only a single protein marker well.
    2. Allow to polymerize for 45 to 60 min at room temperature.
    3. Rinse upper surface of running gel with de-ionized water.
    4. Prepare 2 L of 1x solution of Tank Buffer.
    5. Prepare fresh SDS equilibration buffer (± DTT or Iodoacetamide). 20 ml of buffer is needed for each IPG strip.
    6. Directly after IEF, remove the IPG strip and place it into equilibration tube with plastic backing against the tube wall.
      Note: Handle each strip individually. Don’t mix up the strips.
    7. Add 10 ml of SDS equilibration buffer (+ DTT) to each tube and gently shake on the rocker platform for 10 min at room temperature.
    8. Decant liquid from tube and add 10 ml SDS equilibration buffer (+ DTT) and gently shake on the rocker platform for an additional 15 min at room temperature.
    9. Decant liquid from tube and replace with 10 ml SDS equilibration buffer (+ Iodoacetamide) and gently shake on the rocker platform for 10 min at room temperature.
    10. Decant liquid from tube and add 10 ml SDS equilibration buffer (+ Iodoacetamide) and gently shake on the rocker platform for additional 15 min at room temperature.
    11. Use a tweezer to place IPG strip in-between the glass plates of the gel with gel-side up and acidic end strip (+) nearest the reference well.
    12. Use a 0.75 mm spacer to gently push the IPG strip onto the top surface of the second dimension gel. Avoid bubbles between strip and gel, and ensure good contact.
    13. Seal strip in place with about 500 μl of “cooled” Agarose overlay solution. Let solidify for about 3 min.
    14. Assemble gels into Bio-Rad Protean II.
    15. Fill upper buffer tank with 1x Tank buffer until full. Place remainder in bottom buffer tank with stir bar.
    16. Load 20 μl Bio-Rad protein standards into the reference well.
    17. Electrophorese at 10 mA per gel (constant current) (~ 40-125 V) for about 15 h, or at 20 mA per gel (constant current) (~ 80-250 V) for about 7.5 h. (This is the approximate time required for bromophenol blue dye to migrate within 1 cm of the end of a 10% gel.)

  4. Protein identification from IEF-2DE
    1. Following IEF-2DE protein separation, proteins can be transferred onto PVDF membrane for immunoblot analysis. We used the Bio-Rad semi-dry transfer system and found efficient transfer.
    2. Alternatively, the 2-DE gel can be stained with Coomassie blue to visualize protein spots. Protein spots of interests can be excised from the gel for mass spectrometry analysis following removal of the dye. (Figure 2)


      Figure 2. Total soluble proteins (250 μg) of Arabidopsis thaliana are separated by 2-D gel electrophoresis and identified specific proteins with methionine synthase and lysine-acetylated Abs

Recipes

  1. IEF sample loading buffer                                                                     Final concentration
    Add 0.42 g Urea                                                                                    7 M
    0.15 g Thiourea                                                                                     2 M
    400 μl of 10% CHAPS                                                                           4%
    20 μl of IPG buffer (appropriate pH range for IPG strip)                        2%
    0.01 g DTT                                                                                            1%
    20 μl of 0.1% bromophenol blue                                                            0.002%
    Check that final volume is 1 ml
  2. SDS equilibration buffer (± DTT or Iodoacetamide), make freshly             Final concentration
    Add 1.333 ml of 1.5 M Tris Base (pH 8.8) into a 50 ml tube                       50 mM
    12 ml of 100% glycerol                                                                               30%
    8 ml of 10% SDS                                                                                        2%
    800 μl of 0.1% bromophenol blue                                                               0.002%
    14.41 g Urea                                                                                               6 M
    Add water to 40 ml total volume
    After the solution is completely dissolved. Divide the solution in half
    Add 0.2 g DTT to 20 ml SDS equilibration buffer(and no iodoacetamide)     2%
    Add 0.5 g iodoacetamide to 20 ml SDS equilibration buffer (and no DTT)    5%
  3. 10x Tank buffer                                                                                             Final concentration
    Dissolve 60.4 g Tris Base                                                                               25 mM
    288 g glycine                                                                                                  192 mM
    20 g SDS                                                                                                         0.1%
    In 2 L of water
    Do not adjust pH, 10x pH ~ 8.6, 1x pH ~ 8.1
  4. Agarose overlay solution                                                                   Final Concentration
    980 μl of 1x Tank buffer                                                                     1x
    20 μl of 0.1% bromophenol blue                                                        0.002%
    0.005 g Agarose powder                                                                   0.5%
    Total volume is 1 ml ( ~ 250 μl needed per strip)
    Heat at 95 °C for ~10 min to melt
    Let cool to touch (~70 °C) before use

Acknowledgments

None.

References

  1. Oh, M. H., Clouse, S. D. and Huber, S. C. (2012). Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a post-translational modification and is activated by the juxtamembrane domain. Front Plant Sci 3: 175
  2. Radwan, O., Wu, X., Govindarajulu, M., Libault, M., Neece, D. J., Oh, M. H., Berg, R. H., Stacey, G., Taylor, C. G., Huber, S. C. and Clough, S. J. (2012). 14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation. Plant Physiol 160(4): 2125-2136.
  3. Wu, X., Oh, M. H., Schwarz, E. M., Larue, C. T., Sivaguru, M., Imai, B. S., Yau, P. M., Ort, D. R. and Huber, S. C. (2011). Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis. Plant Physiol 155(4): 1769-1778.

简介

等电聚焦,随后SDS-PAGE(IEF-2DE)在蛋白质pI(蛋白质等电点)和分子量(MW)的二维基质中分离蛋白质。 该技术特别用于区分蛋白质同种型(Radwan等人,2012)和含有翻译后修饰(例如磷酸化)的蛋白质(Oh等人,2012) 和赖氨酸乙酰化(Wu等人,2011)。 通过IEF-2DE分离的蛋白质可以通过使用序列特异性抗体的免疫印迹分析或通过质谱法来鉴定。

材料和试剂

  1. 蛋白质提取物
  2. 苯酚(TE-饱和pH7.9)(Sigma-Aldrich,目录号:P4557)
  3. 0.1M乙酸铵(溶于甲醇)
  4. Tris-base(General Electric Company,目录号:17-1321-01)
  5. 甘氨酸
  6. 尿素(Life Technologies,Invitrogen TM,目录号:ZU10001)
  7. 硫脲(Life Technologies,Invitrogen TM,目录号:ZT10002)
  8. CHAPS(Life Technologies,Invitrogen TM,目录号:ZC10003)
  9. DTT(通用电气公司,目录号:17-1318-01)
  10. 溴酚蓝(General Electric Company,目录号:17-1329-01)
  11. 甘油(General Electric Company,目录号:17-1325-01)
  12. SDS(通用电气公司,目录号:17-1313-01)
  13. 碘乙酰胺(通用电气公司)
  14. 100%乙醇(HPLC级)
  15. 水(HPLC级)
  16. IPG缓冲液(General Electric Company,目录号:17-6000-87)
  17. IPG条(通用电气公司,目录号:17-6000-14)
  18. 固相pH干胶条盖液(General Electric Company,目录号:17-1335-01)
  19. Bradford测定染料试剂(Bio-Rad Laboratories,目录号:500-0006)
  20. SimplyBlue TM SafeStain蛋白染料(Life Technologies,Invitrogen TM,目录号:LC-6065)
  21. Precision Plus TM全部蓝蛋白标准品(Bio-Rad Laboratories,目录号:161-0373)
  22. IEF样本缓冲区(参见配方)
  23. SDS平衡缓冲液(参见配方)
  24. 10x槽缓冲液(见配方)
  25. 琼脂糖覆盖溶液(通用电气公司,目录号:17-0554-01)(参见配方)

设备

  1. 1.5 ml微量离心管
  2. 15 ml管
  3. 等离子聚焦系统(General Electric Company,型号:80-6414-02)
  4. 大型1-D电泳系统(Bio-Rad Laboratories)
  5. 半干转移系统(Bio-Rad Laboratories)
  6. 质谱仪(如MALDI-TOF或串联质谱仪)

程序

  1. 样品准备
    为了通过IEF使蛋白质的分离最大化,蛋白质提取物应当含有最小量的盐和洗涤剂的污染物。 这里是我们用于制备蛋白质提取物(蛋白质提取缓冲液:100 mM Tris-HCl,pH 8.0)进行IEF分析的方法。
    1. 将粗蛋白提取物和冰冷的苯酚(TE-饱和的,pH7.9)以1:1(v:v)比例混合。 涡旋1分钟。
    2. 在4℃下以24,000×g离心混合物15分钟。
    3. 小心地取出上层水相并丢弃(不要打扰相间)。
    4. 向蛋白质混合物中加入等体积的冰冷的50mM Tris-HCl(pH 8.0)。 涡旋1分钟。
    5. 在4℃下以24,000xg离心15分钟。
    6. 取出上层水相并弃去(不要干扰相间)。
    7. 重复步骤4至6一次。
    8. 将低级苯酚相(包括相间)转移到新的冰冷的15ml管中,加入5体积的冰冷的0.1M乙酸铵(在甲醇中)。 涡旋1分钟。
    9. 在-20℃下沉淀蛋白过夜
    10. 轻轻并短暂涡旋以重悬蛋白沉淀。 根据需要在1.5ml微量离心管之间分开样品
    11. 在4℃下以24,000×g离心20分钟,小心地除去上清液。
    12. 向每个微量离心管中加入1ml冰冷的0.1M乙酸铵(在甲醇中)
    13. 让颗粒在缓冲液中"浸泡"10分钟,在-20℃
    14. 在4℃下以24,000×g离心20分钟。
    15. 吸取上清液,小心不要打扰沉淀。
    16. 重复步骤12至15两次。
    17. 用1ml冰冷的100%乙醇进一步洗涤每个沉淀
    18. 在4℃下以24,000×g离心20分钟。
    19. 吸取上清液(尽可能多)。
    20. 用体积约50-100μlIEF样品缓冲液重悬每个蛋白质沉淀。 每个样品的总重悬量为约500μl
    21. 涡旋沉淀,并让其溶解在IEF样品缓冲液在室温下1小时。 不要在IEF样品缓冲液中加热沉淀
    22. 在室温下以24,000×g离心20分钟。 所得上清液适用于IEF分析
    23. 用Bradford测定法定量蛋白质浓度。 将蛋白质提取物储存于-80℃直至使用
  2. IEF蛋白分离
    1. 在室温下解冻蛋白质提取物。
    2. 在24,000×g离心该蛋白质提取物20分钟,在室温下除去任何沉淀。
    3. 取含有200μg总蛋白的等分试样,并用IEF样品缓冲液将体积调节至250μl 注意:13 cm IPG胶条的样品加载量范围大约为100 - 250μg总蛋白。一般来说,我们使用13厘米IPG条(pH 3-10),但我们可以选择条根据蛋白质分析。
    4. 加载250微升的样品均匀地放入陶瓷条支架(13厘米大小)。
    5. 取出13厘米的IPG胶条,并从胶条一侧除去薄塑料密封。
      注意:
      1. 条的pH范围应当与IEF样品缓冲液中的IPG缓冲液的pH范围匹配。
      2. 使用镊子处理IPG胶条。避免接触条的凝胶区域。
    6. 小心地将IPG条带放在陶瓷条支架中的IEF样品表面上,凝胶侧朝下,条带的酸性端(标记为"+")朝向条带支架的尖端(+)端1A)。


      图1.通过蛋白质pI和MW的IEF-2DE蛋白质分离的图示。 A.在第一维中,在IEF条带中蛋白质被蛋白质pI分离。 B. IEF设置显示正侧IEF凝胶应符合陶瓷条支架和IPGphor正电极板的箭头方向。 C.在第二维中,蛋白质在SDS-PAGE中通过蛋白质MW进一步分离
    7. 小心地提升和降低条带,以确保IPG条的凝胶侧与IEF样品溶液良好接触。
    8. 用约1.5ml Immobiline DryStrip Cover Fluid覆盖条。
    9. 用塑料盖盖住条带支架。
    10. 将带状单元放置在IPGphor歧管上,尖头(+)端在大的"+"电极板上。
    11. 开始IEF方案#1(电压辅助的再水合样品加载)20℃,每条50μA,0小时再水化(在非电压下)。
      注意:确保输入正确的IPG条数。
      步骤1 = 30V,360伏电压(Vh)
      步骤2 = 500 V,500 Vh
      步骤3 = 1000 V,1,000 Vh
      步骤4 = 32,000 Vh时为8,000 V
      步骤5,10小时保持在8,000 V
      总运行时间=约20小时(这个电压小时范围推荐用于IPG条的所有pH范围)
    12. IEF分离完成。 良好的停止范围为32,000 Vh至42,000 Vh。
    13. 直接进行IPG条带平衡(如下所述)或将条带存储在密闭容器中,然后在-80℃下继续处理。

  3. SDS-PAGE蛋白分离
    1. 使用仅具有单个蛋白质标记物孔的平梳,倒入1mm×20cm大小的10%丙烯酰胺凝胶(pH 8.8或9.2)。
    2. 允许在室温下聚合45至60分钟。
    3. 用去离子水冲洗运行的凝胶的上表面
    4. 准备2升1x缓冲液槽溶液。
    5. 准备新鲜的SDS平衡缓冲液(±DTT或碘乙酰胺)。每个IPG条需要20ml缓冲液。
    6. 直接在IEF后,取出IPG条,并将其放入平衡管中,用塑料衬垫紧靠管壁 注意:单独处理每个条带。不要混淆条。
    7. 向每个管中加入10ml SDS平衡缓冲液(+ DTT),并在摇床平台上在室温下轻轻摇动10分钟。
    8. 从管中倒出液体,加入10ml SDS平衡缓冲液(+ DTT),并在摇床上在室温下再摇动15分钟。
    9. 从管中倾析液体,并更换为10 ml SDS平衡缓冲液(+碘乙酰胺),并在摇床上在室温下轻轻摇动10分钟。
    10. 从管中倒出液体,加入10ml SDS平衡缓冲液(+碘乙酰胺),在摇床上在室温下再轻轻摇动15分钟。
    11. 使用镊子将IPG条放在凝胶玻璃板之间,凝胶面朝上,最接近参考井的酸性端条(+)。
    12. 使用0.75 mm间隔轻轻推动IPG条带到第二维凝胶的顶部表面。 避免条和凝胶之间出现气泡,并确保良好的接触
    13. 用约500μl"冷却的"琼脂糖覆盖溶液封闭密封条。 让固化约3分钟。
    14. 将凝胶装入Bio-Rad Protean II。
    15. 使用1x储罐缓冲液填充上缓冲罐,直到满罐。 将剩余物置于带搅拌棒的底部缓冲罐中。
    16. 加入20μlBio-Rad蛋白标准品进入参考井
    17. 在每个凝胶(恒定电流)(〜40-125V)下以10mA电泳约15小时,或以每个凝胶20mA(恒定电流)(约80-250V)电泳约7.5小时。 (这是溴酚蓝染料在10%凝胶末端1厘米内迁移所需的大致时间。)

  4. 来自IEF-2DE的蛋白质鉴定
    1. 在IEF-2DE蛋白分离后,可以将蛋白转移到PVDF膜上用于免疫印迹分析。我们使用Bio-Rad半干转移系统,发现有效转移。
    2. 或者,2-DE凝胶可以用考马斯蓝染色以显现蛋白斑点。在除去染料后,可以从凝胶中切除感兴趣的蛋白质点用于质谱分析。 (图2)


      图2.通过2-D凝胶电泳分离拟南芥的总可溶性蛋白(250μg),并用甲硫氨酸合酶和赖氨酸乙酰化Abs鉴定特异性蛋白

食谱

  1. IEF样本加载缓冲区                        ;                         ;                   最终浓度
    加入0.42g尿素                                                                                    7 M
    0.15克硫脲                                                                                   2 M
    将400μl的10%CHAPS                                                                         4%
    20μlIPG缓冲液(IPG条的适当pH范围)                      2%
    0.01 g DTT                                                                                             1%
    20μl的0.1%溴酚蓝                                                         0.002%
    检查最终体积是否为1 ml
  2. SDS平衡缓冲液(±DTT或碘乙酰胺),新鲜制备       最终浓度
    将1.333ml 1.5M Tris碱(pH8.8)加入50ml管中,                     50 mM
    12ml 100%甘油                                                                            30%
    8ml 10%SDS                                                                                      2%
    将800μl的0.1%溴酚蓝                                                            0.002%
    14.41g尿素                                                                                            6 M
    加水至总体积40 ml
    溶液完全溶解后。将解决方案分成两半
    将0.2g DTT加入20ml SDS平衡缓冲液(不含碘乙酰胺)中。    2%
    将0.5g碘乙酰胺加入20ml SDS平衡缓冲液(且不含DTT)中  5%
  3. 10x坦克缓冲区                                                                                         最终浓度
    溶解60.4g Tris碱                                                                            25 mM
    288 g甘氨酸                                                                                               192 mM
    20克SDS                                                                                                       0.1%
    在2升水中
    不要调节pH,10x pH〜8.6,1x pH〜8.1
  4. 琼脂糖重叠解决方案                                                               最终浓度
    980μl1x槽缓冲液                                                                  1x
    20μl的0.1%溴酚蓝                                                     0.002%
    0.005克琼脂糖粉                                                                0.5%
    总体积为1ml(每条需要约250μl)
    在95℃下加热〜10分钟以熔化
    在使用前让触摸(〜70°C)

致谢

没有。

参考文献

  1. Oh,M.H.,Clouse,S.D.and Huber,S.C。(2012)。 BRI1受体激酶的酪氨酸磷酸化通过翻译后修饰发生,并由近膜结构域激活。 Front Plant Sci 3:175
  2. Radwan,O.,Wu,X.,Govindarajulu,M.,Libault,M.,Neece,DJ,Oh,MH,Berg,RH,Stacey,G.,Taylor,CG,Huber,SC和Clough,SJ )。 14-3-3蛋白SGF14c和SGF141在大豆结瘤期间发挥关键作用。 植物生理学 160(4):2125-2136
  3. Wu,X.,Oh,M.H.,Schwarz,E.M.,Larue,C.T.,Sivaguru,M.,Imai,B.S.,Yau,P.M.,Ort,D.R.and Huber, 赖氨酸乙酰化是拟南芥中不同蛋白质的广泛蛋白质修饰。 /a> Plant Physiol 155(4):1769-1778
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 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. Wu, X., Clough, S. J., Huber, S. C. and Oh, M. (2013). IEF-2DE Analysis and Protein Identification. Bio-protocol 3(19): e924. DOI: 10.21769/BioProtoc.924.
  2. Radwan, O., Wu, X., Govindarajulu, M., Libault, M., Neece, D. J., Oh, M. H., Berg, R. H., Stacey, G., Taylor, C. G., Huber, S. C. and Clough, S. J. (2012). 14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation. Plant Physiol 160(4): 2125-2136.
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