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Enzymatic Reactions and Detection of C3 Cleavage Fragments
通过C3被切割后产生的片段检测C3转化酶的活性   

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Abstract

The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body’s most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of ‘single’ proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an example for this kind of enzymatic reactions.

Keywords: Complement system(补体系统), C3(C3), Cathepsin L(组织蛋白酶L), Cleavage(卵裂)

Materials and Reagents

  1. Purified human C3 (Complement Technology, catalog number: A113 )
  2. Purified human C3b (Complement Technology, catalog number: A114 )
  3. Purified human C3a (Complement Technology, catalog number: A118 )
  4. Recombinant human cathepsin L (CTSL) (R&D Systems, catalog number: 952-CY-010 )
  5. 1 M Dithiothreitol (DTT) (Life Technologies, catalog number: P2325 )
  6. Tris acetate gels 3-8% or equivalent (Life Technologies, NuPAGE® Novex®, catalog number: WG1603BOX )
  7. 20x NuPAGE Tris acetate running buffer (Life Technologies, catalog number: LA0041 )
  8. Protein size standard (Life Technologies, catalog number: LC5800 )
  9. 5x protein sample reducing loading buffer (Thermo Fisher Scientific, catalog number: 39000 )
  10. Nitrocellulose membranes (Life Technologies, catalog number: IB301002 )
  11. 5% dried milk powder in 1x PBS (Marvel)
  12. Tween 20 (Sigma-Aldrich, catalog number: P5927 )
  13. Rabbit anti-C3d antibody recognizing the intact and cleaved C3 α-chain (Abcam, catalog number: ab17453 )
  14. Rabbit anti-C3 antibody recognizing the C3 β-chain (MyBioSource, catalog number: MBS857324 )
  15. Mouse anti-C3 antibody recognizing the C3a portion within the uncleaved C3 α-chain (Abcam, catalog number: ab36385 )
  16. Mouse anti-C3a neoepitope antibody recognizing only cleaved C3a (Abcam, catalog number: ab11873 )
  17. Secondary antibodies to rabbit and mouse Ig conjugated to Horseradish peroxidase (HRP) (GE Healthcare, catalog numbers: RPN4301 and NA9310 , respectively)
  18. ClarityTM Western ECL substrate (Bio-Rad Laboratories, catalog number: 170-5060 )
  19. NaCl (Sigma-Aldrich, catalog number S7653 )
  20. KCl (Sigma-Aldrich, catalog number P9333 )
  21. Na2HPO4 (Sigma-Aldrich, catalog number S7907 )
  22. KH2PO4 (Sigma-Aldrich, catalog number P5655 )
  23. MES (free acid) (Sigma-Aldrich, catalogue number M0164 )
  24. Brij35 (Thermo Fisher Scientific, Pierce, catalog number: 28316 )
  25. NaOH (Sigma-Aldrich, catalog number S8045 )
  26. Tris base (Sigma-Aldrich, catalog number: T1503 )
  27. HCl (Sigma-Aldrich, catalog number: 258148 )
  28. 10x phosphate buffered saline (PBS) (see Recipes)
  29. 0.5 M 2- (N-morpholino) ethanesulfonic acid (MES) buffer (see Recipes)
  30. 0.05 M Tris (hydroxymethyl) aminomethan (THAM) buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5427 R )
  2. 37 °C, 5% CO2 cell culture incubator
  3. Heating block (Eppendorf, catalog number: 5382000031 )
  4. Reaction tubes (1.5 ml) (Eppendorf, catalog number: 0030125177 )
  5. Pipettes of several volume sizes (for example, Eppendorf)
  6. Power pack for gel electrophoresis (Bio-Rad Laboratories, catalog number: 164-5070 )
  7. XCell4 SureLockTM Midi-Cell gel running tank (Bio-Rad Laboratories, catalog number: WR0100 )
  8. Western blotting transfer equipment (Life Technologies, iBlot®, catalog number: IB21001 )
  9. Western blot visualization machine Chemi Doc MP imaging system (Bio-Rad Laboratories, catalog number: 170-8280 )
  10. Platform rocker (Stuart, catalog number: R11876-01 )
  11. Cold room
  12. Ice bucket

Software

  1. ImageLab software 4.1 (Bio-Rad Laboratories)

Procedure

  1. Enzymatic cleavage of C3 by cathepsin L (CTSL)
    All reagents are kept on ice at all times unless otherwise stated.
    1. C3 preparation: Human serum-purified C3 is diluted from stock (1 mg/ml) in 1x PBS to a final concentration of 6 μg/ml in 1.5 ml Eppendorf tubes. For example, 3 μl of stock C3 (1 mg/ml) is diluted it in 500 μl 1x PBS to obtain the 6 μg/ml final concentration (see Note 1). This amount of diluted C3 stock is sufficient for 10 enzymatic CTSL cleavage reactions.
    2. Activated cathepsin L (CTSL) preparation (see Note 1): For the activation of CTSL in MES/Brij 35 DTT buffer, DTT (1 M stock) is first diluted 1/200 in 50 mM MES buffer to 5 mM final concentration to generate the activation buffer. MES buffer provides pH of about 6.15 that is optimal for CTSL, while DTT and Brij 35 activate the enzyme. CTSL (stock 200 ng/µl) is then diluted to a final concentration of 40 ng/µl (1: 5) in the activation buffer in 1.5 ml Eppendorf tubes and incubated 15 min on ice without shaking to activate the enzyme (see Note 2). Calculate in advance how many reactions are required for an experiment (see Note 3) and activate the appropriate amount of CTSL.
    3. For a reaction with a ratio of about 1: 1 of C3: CSTL, 50 μl of diluted C3 stock (see step A1) are mixed with 38 μl 1x PBS and 12 μl of activated CTSL (see step A2).
    4. At this step it is recommended to also prepare appropriate control ‘reactions’:
      1. 50 µl of C3 diluted stock + 50 µl 1x PBS into an Eppendorf tube, and
      2. A reaction mixture of 12 μl MES buffer/DTT without CTSL + 38 µl of 1x PBS + 50 μl of C3 diluted stock as control reaction to assess the effect of the CTSL activation buffer without the enzyme on C3.
      3. In addition, similar diluted stocks of 250 ng/100 µl in 1x PBS of C3a and 250 ng/100 µl 1x PBS C3b are prepared and will be used as ‘cleavage controls’.
    5. All reaction mixtures (including controls) are incubated for 30 min (yields complete C3 cleavage) or desired times points at 37 °C in a heating block (alternatively, in a water bath).
    6. Samples are centrifuged at 400 x g for 1 min at room temperature.
    7. Twenty-five microliter of 5x protein sample reducing buffer is added to all samples and the samples then boiled at 95 °C for 3-4 min in preparation for subsequent Western blotting analysis (see section B).
    8. Samples are centrifuged at 400 x g for 1 min at room temperature.
    9. Samples can also be stored at this point for later analysis at -80 °C.

  2. Detection of C3 enzymatic cleavage products by Western blotting
    1. Twenty five microliter of the reaction samples from step A8 [(C3 + CTSL) and controls (appropriately diluted stocks of C3a, C3b, C3 (all three control proteins are first diluted to 6 µg/ml in 1x PBS and then further diluted 1: 1 in 1x PBS) as well as C3 + MES buffer/DTT as described under step A4b)] are loaded along with the protein size marker (10 µl) onto a 3-8% Tris acetated gradient polyacrylamide gel (see Note 4) that had been assembled with 1x NuPAGE Tris acetate running buffer into the Western blotting chamber according to the manufacturer’s protocol (see Note 5).
    2. The loaded protein samples are separated in the gel at 150 V for 60 min or until the front of the dye in the protein sample buffer reaches the bottom end of the gel.
    3. The separated proteins are then transferred onto a nitrocellulose membrane using dry transfer in the Life Technologies iBlot transfer system according to the manufacturer’s protocol. This particular machine does not allow for custom settings and we use the 20 V and 5’ 30’’ transfer conditions (programme 3 on the iBlot machine). If you use another transfer devise, please follow the manufacturer’s instructions.
    4. The nitrocellulose membrane is then placed into a container with enough 5% milk in 1x PBS supplemented with 0.1% Tween 20 to cover the membrane safely and blocked for 1-2 h at room temperature on a rocking shaker (low rotation).
    5. Primary rabbit anti-C3 antibody and rabbit anti-C3d are diluted together at a concentration of 1 μg/ml each in 5% dry milk powder in 1x PBS with 0.1% Tween 20 and incubated with the membrane overnight at 4 °C on a shaker (see Note 6). Ensure that pH remains 7.4 after addition of the milk powder, otherwise adjust accordingly.
    6. The membrane is washed 3 times using 50 ml 1x PBS with 0.1% Tween 20 for 15 min each at room temperature on a shaker.
    7. Secondary anti-rabbit-HRP antibody is diluted in 5% dry milk powder in 1x PBS with 0.1% Tween 20 at 1:2,000 dilution (in enough volume to cover the membrane during shaking) and incubated with the membrane for 2 h at room temperature on a shaker (slow rotation).
    8. The membrane is washed again 3 times using 50 ml 1x PBS with 0.1% Tween 20 for 15 min each at room temperature on a shaker.
    9. The Western blot is developed using the ClarityTM Western ECL Substrate according to the manufacturer’s protocol. Briefly, the two substrate components in the ECL Kit are mixed at a ratio 1:1 and the membrane incubated in the mix for 5 min.
    10. Excess ECL detection fluid is removed from the membrane and the protein bands are then visualized using the Chemi Doc MP imaging system and ImageLab software (see Note 7).


      Figure 1. Cathepsin L cleaves C3 to generate C3b. Serum-purified C3 was incubated with activated Cathepsin L (CTSL) as per protocol for the indicated time points and the reactions terminated by addition of the protein reducing loading buffer. The reaction mixtures where then analysed by Western blotting for cleavage of the C3 α-chain with a mixture of two anti-C3 antibodies recognizing the non-processed α-chain of C3 and also the processed α’-chain of generated C3b (anti-C3d) and the β-chain in both C3 forms (anti-C3). Serum-purified C3 and C3b were loaded as control proteins. Data shown are representative of three (n=3) independently performed experiments.

Notes

  1. Described here is the enzymatic cleavage of C3 by CTSL. If the C3 cleavage products of C3 by an alternative protease by a C3 convertase is to be assessed, please refer to the specific information in regards to activation buffers and suggested enzyme: substrate ratios for those reagents when setting up the experiment. Another note on the buffers used: MES is a buffer which has strongest buffering effect at pH 6.15 where cathepsin L is active. In order to preserve the integrity of papain-like cysteine proteases and to prevent autocatalytic degradation, it has become a general practice to reversibly inactivate stock solutions of cathepsins with methylmethanethiolsulfonate (MMTS) before storage. As a result, addition of a reducing agent such as DTT or an equivalent thiol is required to regenerate the active enzyme prior to use by cleaving the inhibiting S-S bond and freeing the catalytic centre of the enzyme for in vitro activation. Brij 35 concenration at 0.005% does increase cathepsin L activity after 10 min activation at least 20%.
  2. The efficiency of the CTSL activation reaction is variable among different enzyme preparations and vendors, but commonly only about 20%. We determined previously that a ratio of 1:1 - 1:2 of total C3: total CTSL results consistently in 100% C3 cleavage by CTSL within 30 min at 37 °C (Liszewski et al., 2013). These conditions have therefore been chosen in this protocol.
  3. Additional sample preparations may be required if, for example, the inhibition of CTSL-mediated C3 cleavage with inhibitory reagents including chemical inhibitors and blocking antibodies needs to be assessed. Please refer to Liszewski et al. (2013) for the nature and source of such reagents.
  4. It is important to use ‘low percentage’ polyacrylamide gels when the processing of the C3 α-chain is monitored as the molecular weight difference between the α-chain of C3 and the α’-chain of C3b (where the C3a portion is cleaved) is only about 8 kDa. Conversely, when the generation of the cleaved C3a is to be monitored (see Note 5), the usage of higher percentage gels (>12%) or gradient gels (4-20%) is required because of the low molecular weight of 8 kDa of C3a. For more details about size and gel appearance of C3 cleavage products please refer to Nilsson et al. (2011).
  5. This protocol uses the specific Life Technologies/Bio-RAd Midi gel Chamber, the iBlot dry transfer system and the Chemi Doc MP imaging system for different steps in the protocol. Of course, any other system (including semi-dry and wet transfer systems) can be used for the separation and visualization of enzymatic C3 cleavage by Western blotting.
  6. In this protocol (and in Figure 1), the detection of the processing of the α-chain of C3 into the α’-chain of C3b by CTSL is described and shown. For the detection of the cleaved C3a product, the usage of a ‘higher percentage’ or gradient gel is advised (see Note 3) and the antibodies in Step B5 are replaced with a mixture of primary mouse anti-C3 antibody recognizing the C3a portion within the uncleaved C3 α-chain (1:500) and mouse anti-C3a neoepitope antibody recognizing exclusively cleaved C3a (1:1,000).
  7. Detection of both, the CTSL-generated C3b and C3a at the same time can be achieved by using a silver staining protocol after gel separation of the C3 cleavage products in place of the Western blotting protocol (Liszewski et al., 2013). This has the advantage that all C3 cleavage fragments generated during the enzymatic reaction will be detected and not only those recognized by the specific antibodies chosen. However, silver staining is only suitable if purified reagents are used in in vitro rections as decribed here. To detect in vivo/ex vivo generated C3 cleavage by CTSL, for example in cell lysate preparations, silver staining is unsuitable.

Recipes

  1. 10x PBS
    80 g of NaCl
    20 g of KCl
    14.4 g of Na2HPO4
    2.4 g KH2PO4
    Dissolved in 800 ml dH2O and adjust pH to 7.4 with pure HCl under stirring
    Top up with dH2O to 1 L
  2. 0.5 M MES buffer
    Add 97.6 g MES to 800 ml dH2O and dissolve
    Adjust pH to 6.0 with 10 N NaOH and then top up to 1 L with dH2O
    Add 0.5 ml of 10% Brij 35 solution to bring it to 0.005% final concentration
  3. 0.05 M Tris (pH 7.5) buffer (100 ml)
    Mix 0.6 g of Tris base with 80 ml deionized (d) H2O
    Adjust pH to 7.5 with HCl under stirring
    Add dH2O to 100 ml
    Stored at 4 °C

Acknowledgments

This protocol was developed by Claudia Kemper and validated by Martin Kolev. The protocol was originally published in Liszewski et al. (2013). This work was supported by the MRC Research Grant G1002165 (C.K.) the MRC Centre for Transplantation Grant MR/J006742/1, an EU-funded Innovative Medicines Initiative BTCURE (C.K.), and a Wellcome Trust Investigator Award (C.K).

References

  1. Liszewski, M. K., Kolev, M., Le Friec, G., Leung, M., Bertram, P. G., Fara, A. F., Subias, M., Pickering, M. C., Drouet, C., Meri, S., Arstila, T. P., Pekkarinen, P. T., Ma, M., Cope, A., Reinheckel, T., Rodriguez de Cordoba, S., Afzali, B., Atkinson, J. P. and Kemper, C. (2013). Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation. Immunity 39(6): 1143-1157.
  2. Nilsson, U. R., Funke, L., Nilsson, B. and Ekdahl, K. N. (2011). Two conformational forms of target-bound iC3b that distinctively bind complement receptors 1 and 2 and two specific monoclonal antibodies. Ups J Med Sci 116(1): 26-33.

简介

补体组分C3是补体系统的主要效应分子。 C3作为前酶在血液和间质液中循环,并通过酶裂解活化为C3a部分,用作化学引诱物和免疫细胞活化剂的经典过敏毒素和作为机体最有效调理素的C3b部分。 C3切割在大多数情况下由称为C3转化酶的酶复合物介导。 然而,现在越来越清楚的是,通过一系列"单一"蛋白酶将C3切割成生物活性C3a和C3b片段具有高生理学意义。 在这里,我们描述了通过丝氨酸蛋白酶组织蛋白酶L酶切割人C3的方案和通过蛋白质印迹检测裂解产物C3a和C3b作为这种酶反应的实例。

关键字:补体系统, C3, 组织蛋白酶L, 卵裂

材料和试剂

  1. 纯化的人C3(Complement Technology,目录号:A113)
  2. 纯化的人C3b(Complement Technology,目录号:A114)
  3. 纯化的人C3a(补体技术,目录号:A118)
  4. 重组人组织蛋白酶L(CTSL)(R& D Systems,目录号:952-CY-010)
  5. 1M二硫苏糖醇(DTT)(Life Technologies,目录号:P2325)
  6. Tris乙酸盐凝胶3-8%或等同物(Life Technologies,NuPAGE Novex,目录号:WG1603BOX)
  7. 20x NuPAGE Tris乙酸缓冲液(Life Technologies,目录号:LA0041)
  8. 蛋白质大小标准(Life Technologies,目录号:LC5800)
  9. 5x蛋白样品减少加样缓冲液(Thermo Fisher Scientific,目录号:39000)
  10. 硝化纤维素膜(Life Technologies,目录号:IB301002)
  11. 5%奶粉在1×PBS(Marvel)中。
  12. 吐温20(Sigma-Aldrich,目录号:P5927)
  13. 识别完整和切割的C3α链的兔抗C3d抗体(Abcam,目录号:ab17453)
  14. 识别C3β链的兔抗C3抗体(MyBioSource,目录号:MBS857324)
  15. 识别未切割的C3α链中的C3a部分的小鼠抗C3抗体(Abcam,目录号:ab36385)
  16. 仅识别切割的C3a的小鼠抗C3a新表位抗体(Abcam,目录号:ab11873)
  17. 针对偶联到辣根过氧化物酶(HRP)(GE Healthcare,目录号:分别为RPN4301和NA9310)的兔和小鼠Ig的二抗,
  18. Clarity Western ECL底物(Bio-Rad Laboratories,目录号:170-5060)
  19. NaCl(Sigma-Aldrich,目录号S7653)
  20. KCl(Sigma-Aldrich,目录号P9333)
  21. Na 2 HPO 4(Sigma-Aldrich,目录号S7907)
  22. KH sub 2 PO 4(Sigma-Aldrich,目录号P5655)
  23. MES(游离酸)(Sigma-Aldrich,目录号M0164)
  24. Brij35(Thermo Fisher Scientific,Pierce,目录号:28316)
  25. NaOH(Sigma-Aldrich,目录号S8045)
  26. Tris碱(Sigma-Aldrich,目录号:T1503)
  27. HCl(Sigma-Aldrich,目录号:258148)
  28. 10x磷酸盐缓冲盐水(PBS)(见Recipes)
  29. 0.5M 2-(N-吗啉代)乙磺酸(MES)缓冲液(参见配方)。
  30. 0.05 M Tris(羟甲基)氨基甲烷(THAM)缓冲液(参见配方)

设备

  1. 离心机(Eppendorf,型号:5427R)
  2. 37℃,5%CO 2细胞培养箱中培养
  3. 加热块(Eppendorf,目录号:5382000031)
  4. 反应管(1.5ml)(Eppendorf,目录号:0030125177)
  5. 几种体积大小的移液管(例如,Eppendorf)
  6. 用于凝胶电泳的电源组(Bio-Rad Laboratories,目录号:164-5070)
  7. XCell4 SureLock TM Midi-Cell凝胶运行罐(Bio-Rad Laboratories,目录号:WR0100)
  8. Western印迹转移设备(Life Technologies,iBlot ,目录号:IB21001)
  9. Western印迹可视化仪Chemi Doc MP成像系统(Bio-Rad Laboratories,目录号:170-8280)
  10. 平台摇杆(Stuart,目录号:R11876-01)
  11. 冷室
  12. 冰桶

软件

  1. ImageLab软件4.1(Bio-Rad Laboratories)

程序

  1. 组织蛋白酶L(CTSL)对C3的酶裂解
    除非另有说明,所有试剂均保持在冰上。
    1. C3制备:将人血清纯化的C3从1×PBS中的储备液(1mg/ml)稀释至1.5ml Eppendorf管中的6μg/ml的终浓度。例如,将3μl储备液C3(1mg/ml)在500μl1×PBS中稀释,以获得6μg/ml的终浓度(见注1)。这一量的稀释的C3储备足以进行10次酶促CTSL裂解反应。
    2. 活化组织蛋白酶L(CTSL)制剂(参见注释1):为了在MES/Brij 35 DTT缓冲液中激活CTSL,首先将DTT(1M储备液)在50mM MES缓冲液中1/200稀释至5mM终浓度,激活缓冲区。 MES缓冲液提供对于CTSL最佳的约6.15的pH,而DTT和Brij35活化酶。然后在1.5ml Eppendorf管中的活化缓冲液中将CTSL(原液200ng /μl)稀释至40ng /μl(1:5)的终浓度,并在冰上无振荡温育15分钟以活化酶(见注2 )。预先计算实验需要多少反应(见注3),并激活适当数量的CTSL。
    3. 对于C3:CSTL比例为约1:1的反应,将50μl稀释的C3原液(参见步骤A1)与38μl1x PBS和12μl活化的CTSL(参见步骤A2)混合。
    4. 在这一步,建议也准备适当的控制"反应":
      1. 50μlC3稀释液+50μl1×PBS加入到Eppendorf管中,和
      2. 将12μlMES缓冲液/不含CTSL的DTT +38μl1×PBS +50μlC3稀释储液作为对照反应的反应混合物,以评估没有酶的CTSL活化缓冲液对C3的影响。
      3. 此外,制备在1x PBS的C3a和250ng /100μl1×PBS C3b中的250ng /100μl的类似稀释储液,并将其用作"切割对照"。
    5. 将所有反应混合物(包括对照)在37℃下在加热块(或者,在水浴中)温育30分钟(产生完全的C3裂解)或期望的时间点。
    6. 将样品在室温下以400×g离心1分钟。
    7. 向所有样品中加入25微升5x蛋白质样品还原缓冲液,然后将样品在95℃煮沸3-4分钟,以准备随后的Western印迹分析(参见B部分)。
    8. 将样品在室温下以400×g离心1分钟。
    9. 样品也可以在这一点存储,以便以后在-80℃分析
  2. 通过Western印迹检测C3酶裂解产物
    1. 将来自步骤A8 [(C3 + CTSL)和对照(C3a,C3b,C3的适当稀释的储液(所有三种对照蛋白)的二十五微升反应样品首先在1×PBS中稀释至6μg/ml,然后进一步稀释1: 1在1x PBS中)以及如步骤A4b)所述的C3 + MES缓冲液/DTT]与蛋白质大小标记物(10μl)一起装载到3-8%Tris乙酸化的梯度聚丙烯酰胺凝胶上(参见注释4),根据制造商的方案(见注5),用1x NuPAGE Tris乙酸缓冲液装配到Western印迹室中。
    2. 将装载的蛋白质样品在凝胶中在150V下分离60分钟或直到蛋白质样品缓冲液中的染料前端到达凝胶的底端。
    3. 然后根据制造商的方案使用Life Technologies iBlot转移系统中的干转移将分离的蛋白质转移到硝酸纤维素膜上。这台特定的机器不允许自定义设置,我们使用20 V和5'30"的传输条件(iBlot机器上的程序3)。如果使用其他传输设备,请按照制造商的说明进行操作。
    4. 然后将硝酸纤维素膜置于具有在补充有0.1%吐温20的1x PBS中的足够5%牛奶的容器中以安全地覆盖膜并在室温下在摇动摇床上(低旋转)封闭1-2小时。 >
    5. 将初级兔抗C3抗体和兔抗C3d一起以1μg/ml的浓度在具有0.1%Tween 20的1×PBS中的5%干奶粉中稀释,并在4℃下在振荡器上与膜孵育过夜(见附注6)。确保添加奶粉后pH保持7.4,否则进行相应调整。
    6. 使用50ml含有0.1%Tween 20的1x PBS在振荡器上室温下洗膜3次,每次15分钟。
    7. 将第二抗兔-HRP抗体在1%PBS(含有0.1%Tween 20)中的5%奶粉中稀释,以1:2,000稀释(在摇动过程中足以覆盖膜),并在室温下与膜孵育2小时(慢速旋转)。
    8. 将膜再次用50ml含有0.1%Tween 20的1x PBS洗涤3次,每次15分钟,在室温下在振荡器上。
    9. 根据制造商的方案使用Clarity TM Western Western ECL底物开发Western印迹。简言之,将ECL试剂盒中的两种底物组分以1:1的比例混合,将膜在混合物中孵育5分钟。
    10. 从膜上除去过量的ECL检测液,然后使用Chemi Doc MP成像系统和ImageLab软件(参见注释7)显现蛋白条带。


      图1.组织蛋白酶L切割C3以产生C3b。根据所示时间点的方案,将血清纯化的C3与活化的组织蛋白酶L(CTSL)一起孵育,通过加入蛋白质减少负载终止反应缓冲。然后通过蛋白质印迹法分析反应混合物,使用识别C3的未加工的α链的两种抗C3抗体的混合物以及所产生的C3b的加工的α'链(抗-α- C3d)和两种C3形式的β链(抗C3)。加载血清纯化的C3和C3b作为对照蛋白。所示数据代表三个(n = 3)独立进行的实验。

笔记

  1. 这里描述的是CTSL对C3的酶裂解。如果要评估C3通过C3转化酶的替代蛋白酶的C3切割产物,则关于激活缓冲液的具体信息和建议的酶:底物比例,当设置实验时。关于使用的缓冲液的另一个注释:MES是在pH6.15下具有最强缓冲效应的缓冲液,其中组织蛋白酶L是活性的。为了保持木瓜蛋白酶样半胱氨酸蛋白酶的完整性和防止自催化降解,已经成为一般的实践,在储存之前用甲基硫代乙磺酸甲酯(MMTS)可逆地灭活组织蛋白酶的储备溶液。结果,需要加入还原剂如DTT或等同的硫醇以在使用前通过切割抑制性SS键并在体外释放酶的催化中心来再生活性酶。激活。在0.005%浓度下的Brij35浓度在10分钟活化后增加组织蛋白酶L活性至少20%。
  2. CTSL活化反应的效率在不同的酶制剂和供应商中是可变的,但通常只有约20%。我们先前确定了总C 3:总CTSL的1:1-1:2的比率在37℃下30分钟内始终由CTSL在100%C 3裂解中得到(Liszewski等人,2013 )。因此,在本协议中选择了这些条件。
  3. 如果例如需要评估用包括化学抑制剂和阻断抗体的抑制剂抑制CTSL介导的C3切割,则可能需要另外的样品制备。有关此类试剂的性质和来源,请参阅 Liszewski (2013)
  4. 当C3α链的加工被监测为C3的α链和C3b的α'-链之间的分子量差异(其中C3a部分被切割)时,使用"低百分比"聚丙烯酰胺凝胶是重要的,只有约8 kDa。相反,当要监测切割的C3a的产生时(参见注释5),需要使用较高百分比的凝胶(> 12%)或梯度凝胶(4-20%),因为8的低分子量kDa的C3a。关于C3切割产物的大小和凝胶外观的更多细节,请参考Nilsson等人。 (2011)。
  5. 该协议使用特定的Life Technologies/Bio-RAd Midi凝胶室,iBlot干式转移系统和Chemi Doc MP成像系统,用于协议中的不同步骤。当然,任何其他系统(包括半干和湿转移系统)可以用于通过Western印迹分离和显现酶C3裂解。
  6. 在该方案(和图1)中,描述和显示了通过CTSL检测C3的α链进入C3b的α'-链的处理。为了检测裂解的C3a产物,建议使用"较高百分比"或梯度凝胶(参见注释3),并且将步骤B5中的抗体替换为识别C3a部分的初级小鼠抗C3抗体的混合物未切割的C3α链(1:500)和识别仅切割C3a的小鼠抗C3a新表位抗体(1:1000)。
  7. 同时检测CTSL产生的C3b和C3a可以通过使用银染色方案在C3切割产物的凝胶分离之后代替Western印迹方案来实现(Liszewski等人, >,2013)。这具有以下优点:将检测在酶反应期间产生的所有C3切割片段,并且不仅检测由所选择的特异性抗体识别的那些。然而,银染色仅在如在此所述的在体外反应中使用纯化的试剂时才是合适的。为了例如在细胞裂解物制备物中体内/体外检测CTSL产生的C3切割,银染色是不适合的。

食谱

  1. 10x PBS
    80克NaCl
    20克KCl
    14.4g的Na 2 HPO 4
    2.4g KH 2 PO 4 4/
    溶解在800ml dH 2 O中,并在搅拌下用纯HCl将pH调节至7.4。
    充满dH <2> O到1升
  2. 0.5 M MES缓冲区
    将97.6g MES加入到800ml dH 2 O中并溶解
    用10N NaOH调节pH至6.0,然后用dH 2 O补充至1L。
    加入0.5ml 10%Brij 35溶液,使其达到0.005%最终浓度
  3. 0.05M Tris(pH7.5)缓冲液(100ml) 将0.6g Tris碱与80ml去离子(d)H 2 O混合 在搅拌下用HCl调节pH至7.5 将dH <2> O添加到100 ml
    储存在4°C

致谢

该协议由Claudia Kemper开发,并由Martin Kolev验证。 该协议最初发表在Liszewski等人(2013)。 这项工作得到MRC研究基金G1002165(C.K.)MRC移植授权中心MR/J006742/1,欧盟资助的创新药物计划BTCURE(C.K.)和Wellcome Trust Investigator Award(C.K)的支持。

参考文献

  1. 这些研究结果表明,这种方法可以有效地提高产量,提高产量,提高产量,提高产量。 ,Pekkarinen,PT,Ma,M.,Cope,A.,Reinheckel,T.,Rodriguez de Cordoba,S.,Afzali,B.,Atkinson,JP和Kemper, 细胞内补体激活维持T细胞内环境稳定并介导效应细胞分化 免疫 39(6):1143-1157。
  2. Nilsson,U. R.,Funke,L.,Nilsson,B.and Ekdahl,K.N。(2011)。 靶向结合iC3b的两种构象形式,其不同地结合补体受体1和2和两种特异性单克隆抗体 。 Ups J Med Sci 116(1):26-33。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kemper, C. and Kolev, M. (2014). Enzymatic Reactions and Detection of C3 Cleavage Fragments. Bio-protocol 4(16): e1205. DOI: 10.21769/BioProtoc.1205.
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