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Preparation of Parasite Protein Extracts and Western Blot Analysis
寄生虫蛋白质提取物的制备和蛋白印迹分析   

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Abstract

In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available to achieve this. If the protein is present within the parasite or is attached to a cellular structure of the iRBC cell, saponin can be used. This reagent lyses the membranes of infected and uninfected erythrocytes, the Maurer´s clefts (vesicular structures in the iRBC) and the parasitophorous vacuole membrane containing the parasite but leaves the parasite plasma membrane intact, providing a convenient procedure to isolate intact parasites without uRBCs. However, this method has the disadvantage that the host cell cytosol and the parasitophorous vacuole (PV) content of iRBCs are lost. If this has to be avoided, it is possible to use a Percoll gradient to separate intact iRBCs from uRBCs. Sequential treatment with Tetanolysin and saponin can then be used to selectively release the iRBC cytosol and the PV content from the parasite. These selective lysis methods are also suitable to determine the subcellular localisation of a protein of interest.

Keywords: Parasitology(parasitology), Malaria(疟疾), Plasmodium falciparum(恶性疟原虫), Western blotting(Western blot分析)

Materials and Reagents

  1. Parasite culture
    1. Plasmodium falciparum (e.g. clonal line 3D7)
    2. Sterile, human 0+ erythrocyte concentrate (Blood bank)
    3. RPMI complete medium (see Recipes)
      1. RPMI-1640 (AppliChem GmbH, catalog number: A1538,9010 )
      2. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
      3. Glucose (Merck KgaA, catalog number: 1.08342.1000 )
      4. Albumax II (Life Technologies, Gibco®, catalog number: 11021-037 )
      5. Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
      6. 40 mg/ml gentamicine (Ratiopharm)

  2. Parasite protein extraction
    1. Sorbitol (Sigma-Aldrich)
    2. Triton X-114 (Enzo Life Sciences)
    3. 10x PBS (see Recipes)
    4. 0.03 % saponin lysis buffer (Sigma-Aldrich, catalog number: S4521 ) (see Recipes)
    5. Parasite lysis buffer (see Recipes)
    6. Tetanolysin (List Biological Labs, catalog number: 199 ) (see Recipes)
    7. 25x protease inhibitor cocktail mini (Roche Diagnostics, catalog number: 11836170001 ) (see Recipes)
    8. Percoll solutions (GE Healthcare, catalog number: 17-0891-02 ) (see Recipes)

  3. SDS-Page and western blot analysis
    1. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific)
    2. Tris (Merck KGaA)
    3. CAPS (Sigma-Aldrich)
    4. SDS (SERVA Electrophoresis GmbH)
    5. Low fat milk powder (blotting grade) (Carl Roth, catalog number: T145.2 )
    6. ECL solution/Western Blot Detection Kit (Pierce Antibodies)
    7. Antibodies (e.g. mouse anti-GFP, Roche Diagnostics, catalog number: 11814460001 ; horseradish peroxidase-conjugated goat anti-mouse, dianova GmbH, catalog number: 115-035-062 )
    8. Electrophoresis buffer (see Recipes)
    9. 5x SDS sample buffer (see Recipes)
    10. 1 M Tris buffer (pH 6.8) (see Recipes)
    11. 1.5 M Tris buffer (pH 8.8) (see Recipes)
    12. Polyacrylamide gel with 5% stacking gel and 12% separating gel (see Recipes)
    13. CAPS buffer (1 L, 10 mM, pH 11.3) (see Recipes)
    14. Blocking solution (50 ml) (see Recipes)

Equipment

  1. Falcon tubes (15 ml, 50 ml)
  2. Centrifuge
  3. Eppendorf tubes (1.5 ml, 2 ml)
  4. Sterilisation filters (0.22 µm)
  5. Thermo block
  6. Gel electrophoresis chamber (Bio-Rad Laboratories)
  7. Nitrocellulose blotting membrane (Whatman, Protran®)
  8. Chromatography paper (Grade 3 MM CHR) (GE Healthcare)
  9. Tank blot device (Bio-Rad Laboratories)
  10. Rolling device
  11. Transparent sheets
  12. Developer (Agfa-Gevaert Group)
  13. Developing cassette
  14. X-ray film (Agfa-Gevaert Group)

Procedure

Note: All centrifugation steps are carried out at room temperature.

  1. Parasite protein extraction
    1. Saponin lysis to remove the soluble PV content, the host cell cytosol and uRBC.
      1. Centrifuge 10 ml of a Plasmodium falciparum culture (5-10% parasitemia) at 500 x g for 5 min, discard the supernatant and resuspend the pellet in 10 ml 1x PBS. Centrifuge again, resuspend the pellet in 4 ml saponin lysis buffer (0.03%) and incubate on ice for 5-20 min.
      2. Centrifuge the lysate at 16,000 x g for 5 min to pellet the parasites and repeatedly wash the pellet with 1x PBS until the supernatant shows no red colour anymore.
        Note: Usually 3 washing steps are necessary.
      3. Discard the supernatant and add 2-8 µl 25x protease inhibitor cocktail. Depending on the size of the parasite pellet, resuspend the pellet in 50-200 µl parasite lysis buffer.
        Note: In general 100 µl parasite lysis buffer will work for 10 ml of a parasite culture >5% parasitemia but this depends on the proportion of schizont stage parasites as these stages contain a lot of DNA. Addition of DNase can be used to reduce the viscosity of the extract.
      4. The extracts can be stored at -20 °C if not used immediately for SDS-PAGE.
    2. Percoll gradient to obtain intact iRBC followed by saponin lysis to separate soluble host cell and PV content from parasite material.
      1. Centrifuge 10 ml of a Plasmodium falciparum culture (5-10% parasitemia) at 500 x g for 5 min, discard the supernatant and resuspend the pellet in 10 ml 1x PBS. Centrifuge again and remove the supernatant.
      2. Prepare the percoll gradient by adding first 500 µl 80% percoll solution to a 2 ml tube, then carefully layering 500 µl 60% percoll solution on top of the 80% percoll and finally layering 500 µl 40% percoll solution on top of the 60% percoll (Figure 1, Percoll gradient).
        Note: Use the gradient immediately.
      3. Resuspend the pellet with 200 µl 1x PBS and slowly pipette the parasite solution on top of the gradient. Centrifuge immediately at 16,000 x g for 5 min.
        Note: Touch the side of the tube with the pipette tip while adding the parasites to avoid mixing of the solutions.
      4. Carefully collect the desired parasite stage band into a new 1.5 ml tube (see Figure 1 for selection of correct phase from the gradient) and add 1 ml 1x PBS.
        Note: If too much percoll solution is transferred, for instance if no clear iRBC phase was apparent (Figure 1A, scenario 3), the parasites may not pellet properly after the first washing step. In this case only remove the top region after centrifugation of the first wash, and replace again with 1x PBS to bring down the overall percoll concentration of the solution. The iRBCs should then pellet after in the next centrifugation step.
      5. Centrifuge at 16,000 x g for 5 min and wash the parasite pellet at least 3x with 1 ml 1x PBS.
        Note: Use a fresh tube for each washing step.
      6. Resuspend the pellet in 50 µl PBS, then add 50 µl saponin lysis buffer (0.03%) and incubate on ice for 5 min.
      7. Centrifuge the lysate at 16,000 x g for 5 min. Transfer the saponin supernatant into a fresh tube (final extract of host cell cytosol and PV content) and add 4 µl 25x protease inhibitor cocktail. The extract can be stored at -20 °C if not used immediately for SDS-PAGE.
      8. Wash the pellet with 1x PBS until the supernatant is clear.
        Note: In general 3 washing steps are necessary.
      9. Add 4 µl 25x protease inhibitor cocktail to the pellet. Resuspend the pellet in 100 µl parasite lysis buffer. The extract can be stored at -20 °C if not used immediately for SDS-PAGE.


      Figure 1. Percoll gradient and outcomes. A. Preparation/Set-up of a Percoll gradient without and with parasite culture (top left). Three possible results after centrifugation are shown under 'outcomes' (top right): 1) one band consisting of mostly debris, merozoites and segmented schizonts at the border between the 40% Percoll and the aqueous region (present above the 40% percoll after centrifugation) and one band above uRBC/ring stage region consisting of younger schizonts and trophozoites (This is the fraction to harvest.); 2) similar appearance to 1) but with multiple bands below the debris/merozoites/segmented schizonts phase; these bands usually consist of different stages of iRBCs and can all be collected; 3) no clearly defined band; in this case collect the indicated fraction (from the mid 40% to the region just above the uRBCs and ring stage phase, making sure not to disturb this phase). Although in this case the stages may be too distributed to be seen as a clear band in the gradient, this usually will still result in a good recovery once pelleted and washed. B. Example of a purification of iRBCs infected with PF13_0191-GFP parasites. GFP, PF13_0191-GFP fluorescence; DIC, differential interference contrast; merge, both images merged.

    3. Percoll gradient and sequential treatment with tetanolysin and saponin to obtain extracts of the host cell cytosol and the PV content, respectively.
      1. Perform steps A2a-e.
      2. Resuspend the parasite pellet in 99 µl 1x PBS and add 1 µl tetanolysin (1 µg/ml). Mix the solution immediately by flipping the tube with your finger. Incubate at 37 °C for 30 min.
        Note: The tetanolysin activity may vary with batch and depending on the number of freeze thaw cycles of the aliquot used. If standardised results are needed, activity testing with uRBCs can be conducted before each use. For this incubate serial dilutions of tetanolysin with 20 µl of 70% haematocrit RBCs for 1 min at 37 °C followed immediately by centrifugation at 16,000 x g for 3 min to assess lysis (pellet of uRBCs left). For the percoll pellet use 10x the amount of tetanolysin required to just lyse all of the 20 µl uRBCs. Note that the actual number of parasites obtained after percoll purification also influences lysis efficiency.
      3. Centrifuge the lysate at 16,000 x g for 5 min. Transfer the tetanolysin supernatant into a fresh tube (final extract of host cell cytosol) and add 4 µl 25x protease inhibitor cocktail. The extract can be stored at -20 °C if not used immediately for SDS-PAGE.
      4. Wash the pellet with 1x PBS until the supernatant is clear.
      5. Resuspend the pellet in 50 µl 1x PBS, add 50 µl saponin lysis buffer and incubate on ice for 5 min.
      6. Perform steps A2g-i.

  2. SDS-PAGE and western blot analysis of parasite protein extracts
    1. SDS-page
      1. Prepare a polyacrylamide gel with a 5% stacking and a 12% separating gel, 0.75 mm thickness.
      2. Centrifuge the (thawed) parasite protein extract at 16,000 x g for 5 min and transfer the supernatant into a fresh tube.
      3. Add the required amount of 5x SDS sample buffer and incubate at 95 °C for 5 min.
        Note: Some multi-transmembrane proteins can aggregate if heated, warming the extract to 50 °C may then be applied.
      4. Load the prestained protein ladder (4 µl) and 8 µl of the extract into the slots of the gel in the electrophoresis chamber filled with electrophoresis buffer and run the gel at 200 V for 1 h.
    2. Western blot analysis
      1. Soak a nitrocellulose membrane and 6 sheets of chromatography paper cut to the size of the gel in 10 mM CAPS (pH 11.2).
      2. Prepare the blot in the following order: anode (+), sponge, 3 sheets of chromatography paper, the nitrocellulose membrane, the SDS gel, 3 sheets of chromatography paper, sponge, cathode (-).
      3. Perform protein transfer in a tank blot device at 15 V per gel and 4 °C over night.
      4. Dismantle the blot-sandwich and immediately transfer membrane to 1x PBS for a brief rinse.
        Note: Gel can be stained with Coomassie to check efficient transfer.
      5. Incubate the membrane with 10 ml blocking solution in a 50 ml falcon tube at room temperature for 1 h on a rolling device.
      6. Replace blocking solution with 5 ml of fresh blocking solution. Add 5 µl primary antibody (e.g. mouse anti-GFP 1:1,000) to the blocking solution and incubate the membrane at room temperature for 3 h on a rolling device.
      7. Wash the membrane 5x with 10-20 ml 1x PBS at room temperature for 5 min on a rolling device.
      8. Dilute 1.7 µl secondary antibody (e.g. horseradish peroxidase-conjugated goat anti-mouse 1:3,000) in 5 ml blocking solution and incubate the membrane at room temperature for 1 h on a rolling device.
      9. Wash the membrane 5x with 10-20 ml 1x PBS at room temperature for 5 min on a rolling device.
      10. Incubate the membrane with enough ECL solution to cover the blot (1.5-3 ml) at room temperature for 5 min on a rolling device.
      11. Transfer the membrane between 2 layers of transparent sheets. Apply X-ray films for different times in the dark.
      12. Develop the X-ray films in the developer.

Recipes

  1. RPMI complete medium (1 L)
    15.87 g RPMI-1640
    1 g NaHCO3
    2 g glucose
    5 g Albumax II
    0.0272 g hypoxanthine
    0.5 ml gentamicine
    Adjust pH to 7.2 with HCl
    Add dH2O to 1 L
    Filter sterilize (0.22 µm) and stored at 4 °C
  2. 10x PBS (1 L)
    5.7 g Na2HPO4
    1.25 g NaH2PO4
    15.2 g NaCl
    Adjust pH to 7.4
    Add dH2O to 1 L
  3. 0.03% saponin lysis buffer (50 ml)
    Mix 0.0015 g saponin with 50 ml 1x PBS
    Stored at 4 °C for up to 1 month
  4. Parasite lysis buffer (10 ml)
    4 ml 10% SDS (4 % final)
    0.5 ml 10% Triton X-114
    5 ml 1x PBS
    0.5 ml dH2O
  5. Tetanolysin
    Dissolved at 1 µg/µl in dH2O
    Make aliquots
  6. 25x protease inhibitor cocktail mini
    Dissolve 1 tablet in 400 µl dH2O and stored aliquots at -20 °C
  7. Percoll solutions
    90% (50 ml): mix 45 ml Percoll with 5 ml 10x PBS
    80% (10 ml): mix 0.8 g sorbitol with 8.9 ml 90% Percoll and 1.1 ml RPMI complete medium
    60% (10 ml): mix 0.8 g sorbitol with 6.7 ml 90% Percoll and 3.3 ml RPMI complete medium
    40% (10 ml): mix 0.8 g sorbitol with 4.4 ml 90% Percoll and 5.6 ml RPMI complete medium
    Filter sterilize (0.22 µm) and stored at 4 °C
  8. Electrophoresis buffer (1 L)
    3.03 g Tris
    14.4 g glycine
    1 g SDS
    Add dH2O to 1 L
  9. 5x SDS sample buffer (50 ml)
    1.82 g TrisHCl (pH 6.8)
    5 g SDS
    25 ml glycerol
    3.86 g DTT
    0.05% bromophenol blue
    Add dH2O to 50 ml
  10. 1 M Tris buffer (pH 6.8) (1 L)
    Mix 121.1 g Tris with 800 ml dH2O
    pH to 6.8 with HCl and add dH2O to 1 L
  11. 1.5 M Tris buffer (pH 8.8) (1 L)
    181.65 g Tris with 800 ml dH2O
    pH to 8.8 with HCl and add dH2O to 1 L
  12. 5% stacking gel (2 gels)
    0.75 ml 1 M Tris (pH 6.8)
    4.35 ml dH2O
    0.75 ml 40% acrylamide
    6 µl TEMED
    60 µl 10% SDS
    60 µl 10% APS
  13. 12% separating gel (2 gels)
    2.5 ml 1.5 M Tris (pH 8.8)
    4.2 ml dH2O
    3 ml 40% acrylamide
    4 µl TEMED
    100 µl 10% SDS
    100 µl 10% APS
  14. CAPS buffer (1 L, 10 mM, pH 11.3)
    2.21 g CAPS in 800 ml dH2O
    pH to 11.3 with sodium hydroxide and add dH2O to 1 L
  15. Blocking solution (50 ml)
    Mix 2.5 g milk powder with 50 ml 1x PBS
    Keep at 4 °C

Acknowledgments

The use of pore-forming toxins in P. falciparum was pioneered by the Lingelbach lab: Ansorge et al. (1996). The Percoll gradient is a modified version of the protocol from Aley et al. (1986).

References

  1. Aley, S. B., Sherwood, J. A., Marsh, K., Eidelman, O. and Howard, R. J. (1986). Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients. Parasitology 92 (Pt 3): 511-525.
  2. Ansorge, I., Benting, J., Bhakdi, S. and Lingelbach, K. (1996). Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O. Biochem J 315 (Pt 1): 307-314.
  3. Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., Kadekoppala, M., Hecht, L., Holder, A. A., Gilberger, T. W. and Spielmann, T. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.

简介

为了制备用于蛋白质印迹分析的恶性疟原虫血液阶段的蛋白质提取物,需要将感染的红细胞(iRBC)与构成寄生虫的主体的未感染的红细胞(uRBC)分离文化。根据感兴趣的寄生虫蛋白的定位,可以使用不同的方法来实现这一点。如果蛋白质存在于寄生虫内或附着于iRBC细胞的细胞结构,则可以使用皂苷。该试剂裂解感染的和未感染的红细胞的膜,Maurer's裂口(iRBC中的囊泡结构)和含有寄生虫的寄生虫液泡膜,但是使寄生虫质膜完整,提供了一种方便的方法来分离没有uRBC的完整寄生虫。然而,该方法的缺点是iRBC的宿主细胞胞质和寄生泡(PV)含量丢失。如果这必须避免,可以使用Percoll梯度将完整的iRBC与uRBC分离。然后可以使用Tetanolysin和皂苷的顺序处理来从寄生虫选择性释放iRBC细胞溶质和PV含量。这些选择性裂解方法也适合于确定目标蛋白质的亚细胞定位

关键字:parasitology, 疟疾, 恶性疟原虫, Western blot分析

材料和试剂

  1. 寄生虫培养
    1. 恶性疟原虫(例如克隆系3D7)
    2. 无菌,人类0 + 红细胞浓缩物(血库)
    3. RPMI完全培养基(见配方)
      1. RPMI-1640(AppliChem GmbH,目录号:A1538,9010)
      2. NaHCO 3(Sigma-Aldrich,目录号:S5761)
      3. 葡萄糖(Merck KgaA,目录号:1.08342.1000)
      4. Albumax II(Life Technologies,Gibco ,目录号:11021-037)
      5. 次黄嘌呤(Sigma-Aldrich,目录号:H9636)
      6. 40 mg/ml庆大霉素(Ratiopharm)

  2. 寄生虫蛋白提取
    1. 山梨醇(Sigma-Aldrich)
    2. Triton X-114(Enzo Life Sciences)
    3. 10x PBS(请参阅配方)
    4. 0.03%皂苷裂解缓冲液(Sigma-Aldrich,目录号:S4521)(参见Recipes)
    5. 寄生虫裂解缓冲液(见配方)
    6. Tetanolysin(List Biological Labs,目录号:199)(参见Recipes)
    7. 25x蛋白酶抑制剂混合物迷你(Roche Diagnostics,目录号:11836170001)(参见Recipes)
    8. Percoll溶液(GE Healthcare,目录号:17-0891-02)(参见Recipes)

  3. SDS-PAGE和western印迹分析
    1. PageRuler预染蛋白梯子(Thermo Fisher Scientific)
    2. Tris(Merck KGaA)
    3. CAPS(Sigma-Aldrich)
    4. SDS(SERVA Electrophoresis GmbH)
    5. 低脂奶粉(印迹级)(Carl Roth,目录号:T145.2)
    6. ECL溶液/Western印迹检测试剂盒(Pierce抗体)
    7. 抗体(例如小鼠抗GFP,Roche Diagnostics,目录号: 11814460001; 辣根过氧化物酶缀合的山羊抗小鼠,dianova GmbH,目录号:115-035-062)
    8. 电泳缓冲液(参见配方)
    9. 5x SDS样品缓冲液(见配方)
    10. 1 M Tris缓冲液(pH 6.8)(参见配方)
    11. 1.5 M Tris缓冲液(pH 8.8)(参见配方)
    12. 具有5%堆积凝胶和12%分离凝胶的聚丙烯酰胺凝胶(参见配方)
    13. CAPS缓冲液(1L,10mM,pH 11.3)(参见配方)
    14. 封闭溶液(50ml)(见配方)

设备

  1. Falcon管(15ml,50ml)
  2. 离心机
  3. Eppendorf管(1.5ml,2ml)
  4. 灭菌过滤器(0.22μm)
  5. 热块
  6. 凝胶电泳室(Bio-Rad Laboratories)
  7. 硝酸纤维素印迹膜(Whatman,Protran )
  8. 色谱纸(3级MM CHR)(GE Healthcare)
  9. 罐印迹装置(Bio-Rad Laboratories)
  10. 滚动装置
  11. 透明纸张
  12. 开发人员(Agfa-Gevaert Group)
  13. 开发录音带
  14. X光胶片(Agfa-Gevaert Group)

程序

注意:所有离心步骤均在室温下进行。

  1. 寄生虫蛋白提取
    1. 皂苷裂解除去可溶性PV含量,宿主细胞胞质溶胶和uRBC。
      1. 离心10毫升恶性疟原虫培养物(5-10% 寄生虫血症)以500×g离心5分钟,弃去上清液并重悬   在10ml 1x PBS中的沉淀。 再次离心,在4中重悬沉淀   ml皂苷裂解缓冲液(0.03%),并在冰上孵育5-20分钟
      2. 在16,000×g离心裂解物5分钟以沉淀寄生虫 并用1×PBS反复洗涤沉淀,直至上清液显示 没有红色了。
        注意:通常需要执行3个清洗步骤。
      3. 弃去上清液,加入2-8μl25x蛋白酶抑制剂 鸡尾酒。 根据寄生虫颗粒的大小,重悬 沉淀在50-200μl寄生虫裂解缓冲液中。
        注意:一般100μl 寄生虫裂解缓冲液将用于10ml> 5%的寄生虫培养物, 寄生虫,但这取决于裂殖体阶段的比例 寄生虫作为这些阶段包含大量的DNA。 加入DNase即可   用于降低提取物的粘度。
      4. 如果不立即用于SDS-PAGE,提取物可以储存在-20℃。
    2. Percoll梯度以获得完整的iRBC,随后皂甙裂解 单独的可溶性宿主细胞和PV含量与寄生虫材料。
      1. 离心10毫升恶性疟原虫培养物(5-10% 寄生虫血症)以500×g离心5分钟,弃去上清液并重悬   在10ml 1x PBS中的沉淀。 再次离心并取出 上清液
      2. 通过添加前500个准备percoll渐变 μl80%percoll溶液到2ml试管中,然后仔细分层500μl 60%percoll溶液在80%percoll顶部,最后分层 500μl40%percoll溶液在60%percoll顶部(图1, Percoll梯度)。
        注意:立即使用渐变。
      3. 用200μl1×PBS重悬沉淀,并缓慢吸取寄生虫   溶液在梯度顶部。 立即以16,000×g离心5分钟。
        注意:在添加寄生虫时,用移液器尖端触摸管的侧面,以避免溶液混合。
      4. 小心地收集所需的寄生虫阶段带到一个新的1.5毫升 (参见图1,用于从梯度中选择正确的相)和   加入1ml 1×PBS。
        注意:如果太多的percoll溶液被转移,   例如如果没有明显的iRBC阶段(图1A,情景 3),在第一洗涤步骤后寄生虫可能不会适当地沉淀。 在这种情况下,只有在离心后才去除顶部区域 首先洗涤,并再次用1x PBS代替整体 percoll浓度的溶液。 iRBC应该沉淀 在下一次离心步骤后。
      5. 以16,000×g离心5分钟,并用1ml 1×PBS洗涤寄生虫沉淀至少3次。
        注意:每个洗涤步骤都使用新鲜管。
      6. 将沉淀重悬在50μlPBS中,然后加入50μl皂苷裂解缓冲液(0.03%),并在冰上孵育5分钟。
      7. 在16,000×g离心裂解物5分钟。 转移皂苷 上清液置于新管中(宿主细胞细胞溶质和PV的最终提取物   内容物)并加入4μl25x蛋白酶抑制剂混合物。 提取物可以 如果不立即用于SDS-PAGE,则储存在-20℃
      8. 用1×PBS洗涤沉淀,直至上清液澄清 注意:通常需要执行3个清洗步骤。
      9. 加入4微升25x蛋白酶抑制剂混合物到沉淀。 重新悬挂 沉淀在100μl寄生虫裂解缓冲液中。 提取物可以在-20℃保存   °C,如果不立即用于SDS-PAGE

      图1. Percoll渐变 和结果。 A.准备/设置Percoll渐变没有和 与寄生虫培养(左上)。后三种可能的结果 离心显示在"结果"(右上):1)一条带 主要由碎片,裂殖子和分段裂片组成 40%Percoll和水性区域之间的边界(存在于  离心后40%percoll)和uRBC /环阶段上方的一条带 区域由更年轻的裂殖体和滋养体组成(这是 分数收获。 2)外观类似于1)但具有多个 碎片/裂殖子/分段裂殖相下的带;这些频段  通常由不同阶段的iRBC组成,并且可以全部收集; 3)没有明确界定的带;在这种情况下收集指示的部分 (从中间40%到刚好在uRBC和环状阶段上方的区域 相,确保不干扰此阶段)。虽然在这种情况下  阶段可能太分散以致不能看作是清楚的带 梯度,这通常仍然会导致一次好的恢复 沉淀并洗涤。 B.用i。感染的iRBC的纯化的实施例  PF13_0191-GFP寄生虫。 GFP,PF13_0191-GFP荧光; DIC, 微分干涉对比;合并,两个图像合并。

    3. Percoll梯度和连续用溶组织蛋白酶和皂苷处理   以获得宿主细胞胞质溶胶和PV含量的提取物, 分别。
      1. 执行步骤A2a-e。
      2. 重新悬挂 寄生虫沉淀在99μl1×PBS中,并加入1μl溶组蛋白溶酶(1μg/ml)。 用手指翻转管子立即混合溶液。 在37℃孵育30分钟。
        注意:溶支原体活性可能不同   与批次和取决于冻融循环的次数 使用等分试样。 如果需要标准化结果,活动测试用 uRBCs可以在每次使用前进行。 对于这个孵化系列 溶组织素溶液用20μl70%血细胞比容RBCs稀释1分钟   37℃,然后立即以16,000×g离心3分钟至   评估裂解(剩余的uRBCs沉淀)。 对于percoll丸使用10x   只溶解所有20μluRBC所需的溶胀素的量。 注意,在percoll后获得的寄生虫的实际数量 纯化也影响裂解效率。
      3. 离心机 裂解物以16,000×g离心5分钟。 转移溶组织蛋白酶上清液 到新管(宿主细胞细胞溶质的最终提取物),并加入4μl25x 蛋白酶抑制剂混合物。 如果没有,提取物可以储存在-20℃ 立即用于SDS-PAGE。
      4. 用1×PBS洗涤沉淀,直至上清液澄清
      5. 将沉淀重悬于50μl1x PBS中,加入50μl皂苷裂解缓冲液,在冰上孵育5分钟。
      6. 执行步骤A2g-i。

  2. 寄生虫蛋白提取物的SDS-PAGE和western印迹分析
    1. SDS页
      1. 准备一个聚丙烯酰胺凝胶与5%堆叠和12%分离凝胶,0.75毫米厚度
      2. 将(解冻的)寄生虫蛋白质提取物以16,000×g离心5分钟,并将上清液转移到新管中。
      3. 加入所需量的5×SDS样品缓冲液,并在95°C孵育5分钟 注意:如果加热,一些多跨膜蛋白可以聚集,然后可以将提取物温热至50℃。
      4. 加载预染蛋白梯(4微升)和8微升的提取物   电泳室中凝胶的狭槽充满 电泳缓冲液,并在200V下运行凝胶1小时。
    2. 蛋白质印迹分析
      1. 浸泡硝酸纤维素膜和6片色谱纸,切成10mM CAPS(pH 11.2)中的凝胶大小。
      2. 按以下顺序准备印迹:阳极(+),海绵,3页 的层析纸,硝酸纤维素膜,SDS凝胶,3 色谱纸,海绵,阴极( - )。
      3. 在罐式印迹装置中以15V /凝胶和4℃过夜进行蛋白质转移
      4. 拆除印迹夹心,立即将膜转移到1x PBS进行短暂冲洗。
        注意:凝胶可以用考马斯染色以检查有效转移。
      5. 在室温下,在滚动装置上,在50ml Falcon管中用10ml封闭溶液孵育膜1小时。
      6. 用5ml新鲜封闭溶液替换封闭溶液。 添加5 μl第一抗体(例如小鼠抗GFP 1:1,000)至阻断 溶液,并在室温下将膜孵育3小时 滚动装置
      7. 用10-20ml 1×PBS在室温下在滚动装置上洗涤膜5x5分钟
      8. 稀释1.7μl第二抗体(例如辣根) 过氧化物酶缀合的山羊抗小鼠1:3,000)的5ml封闭溶液中   并在室温下在滚动上孵育膜1小时 设备。
      9. 用10-20ml 1×PBS在室温下在滚动装置上洗涤膜5x5分钟
      10. 孵育膜与足够的ECL溶液覆盖的印迹 (1.5-3ml)在室温下在滚动装置上5分钟
      11. 将膜转移到2层透明片材之间。 在黑暗中适用于不同时间的X光胶片。
      12. 在开发者中开发X射线胶片。

食谱

  1. RPMI完全培养基(1L)
    15.87g RPMI-1640
    1g NaHCO 3 3/h 2 g葡萄糖 5克Albumax II
    0.0272g次黄嘌呤 0.5 ml庆大霉素
    用盐酸调节pH至7.2 将dH <2> O添加到1 L
    过滤灭菌(0.22μm)并在4℃下保存
  2. 10x PBS(1L)
    5.7g Na 2 HPO 4。 1.25g NaH 2 PO 4 sub/d 15.2g NaCl
    将pH调节至7.4
    将dH <2> O添加到1 L
  3. 0.03%皂苷裂解缓冲液(50ml) 将0.0015g皂苷与50ml 1x PBS混合
    储存于4°C长达1个月
  4. 寄生虫裂解缓冲液(10ml) 4ml 10%SDS(4%最终) 0.5ml 10%Triton X-114 5 ml 1x PBS
    0.5ml dH 2 O 2 /
  5. 溶血素 在dH 2 O中溶解为1μg/μl
    制作等份
  6. 25x蛋白酶抑制剂鸡尾酒
    将1片溶于400μldH 2 O中溶解,并储存于-20℃下。
  7. Percoll溶液
    90%(50ml):将45ml Percoll与5ml 10×PBS混合物
    80%(10ml):将0.8g山梨醇与8.9ml 90%Percoll和1.1ml RPMI完全培养基混合
    60%(10ml):将0.8g山梨醇与6.7ml 90%Percoll和3.3ml RPMI完全培养基混合
    40%(10ml):将0.8g山梨醇与4.4ml 90%Percoll和5.6ml RPMI完全培养基混合
    过滤灭菌(0.22μm)并在4℃下保存
  8. 电泳缓冲液(1 L)
    3.03g Tris
    14.4g甘氨酸
    1克SDS
    将dH <2> O添加到1 L
  9. 5×SDS样品缓冲液(50ml) 1.82g TrisHCl(pH 6.8)
    5克SDS
    25ml甘油 3.86 g DTT
    0.05%溴酚蓝
    将dH <2> O添加到50ml
  10. 1M Tris缓冲液(pH6.8)(1L) 将121.1g Tris与800ml dH 2 O混合 用HCl将pH调节至6.8,并将dH 2 O加至1L
  11. 1.5M Tris缓冲液(pH8.8)(1L) 181.65g Tris与800ml dH 2 O
    用HCl将pH调至8.8,并将dH 2 O加至1L
  12. 5%堆积凝胶(2凝胶) 0.75ml 1M Tris(pH6.8)
    4.35ml dH 2 O·dm / 0.75ml 40%丙烯酰胺 6μlTEMED
    60μl10%SDS
    60微升10%APS
  13. 12%分离凝胶(2块凝胶) 2.5ml 1.5M Tris(pH8.8)
    4.2ml dH 2 O 2 / 3ml 40%丙烯酰胺 4μlTEMED
    100μl10%SDS
    100μl10%APS
  14. CAPS缓冲液(1L,10mM,pH 11.3) 在800ml dH 2 O中的2.21g CAPS 用氢氧化钠将pH调至11.3,并将dH 2 O加至1L
  15. 封闭溶液(50ml)
    将2.5g奶粉与50ml 1x PBS混合,
    保持在4°C

致谢

在p中使用成孔毒素。 falciparum 是由Lingelbach实验室率先开发的:Ansorge等人(1996)。 Percoll梯度是来自Aley等人的方案的修改版本(1986)。

参考文献

  1. Aley,S.B.,Sherwood,J.A.,Marsh,K.,Eidelman,O。和Howard,R.J。(1986)。 鉴定山梨醇富集的恶性疟原虫感染的红细胞上的分离物特异性蛋白 来自冈比亚病人。寄生虫学 92(第3篇):511-525。
  2. Ansorge,I.,Benting,J.,Bhakdi,S。和Lingelbach,K。(1996)。 在恶性疟原虫感染的红细胞中透过细胞透化的红细胞中的蛋白质分选 形成蛋白链球菌溶血素O. 生物化学杂志315(第1篇):307-314。
  3. Heiber,A.,Kruse,F.,Pick,C.,Gruring,C.,Flemming,S.,Oberli,A.,Schoeler,H.,Retzlaff,S.,Mesen-Ramirez,P.,Hiss,JA ,Kadekoppala,M.,Hecht,L.,Holder,AA,Gilberger,TW和Spielmann,T。(2013)。 新PNEP的鉴定表明实质的非PEXEL exportome并且支持恶性疟原虫的常见特征 蛋白质导出。 PLoS Pathog 9(8):e1003546。
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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: © 2014 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. Heiber, A. and Spielmann, T. (2014). Preparation of Parasite Protein Extracts and Western Blot Analysis. Bio-protocol 4(11): e1136. DOI: 10.21769/BioProtoc.1136.
  2. Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., Kadekoppala, M., Hecht, L., Holder, A. A., Gilberger, T. W. and Spielmann, T. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.
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