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Cell Surface Protein Biotinylation and Analysis
细胞表面蛋白质的生物素修饰和分析   

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参见作者原研究论文

本实验方案简略版
PLOS ONE
Jan 2013

Abstract

A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control).

Keywords: Cell surface protein biotinylation (细胞表面蛋白的生物素化), Sds-page electrophoresis (SDS-PAGE (同时修改英文?)), Western blotting (蛋白质印迹法), Membrane proteins (膜蛋白)

Materials and Reagents

  1. Human embryonic kidney HEK293 cells (Health Protection Agency Culture Collections, catalog number: 96121229 )
  2. Biotin reagent: EZ-link Sulfo-NHS-LC-LC-biotin (Thermo-Pierce, catalog number: 21338 )
  3. Electrophoresis grade Glycine (Sigma-Aldrich, catalog number: G8898 )
  4. Immobilized/NeutrAvidin Ultralink Resin (Thermo-Pierce, catalog number: 53150 )
  5. PierceTM BCA Protein Assay Kit (Thermo-Pierce, catalog number: 23227 )
  6. Polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, catalog number: RPN303F )
  7. Nonfat dry milk (AppliChem GmbH, catalog number: A0830 )
  8. Chemiluminescence substrate: Super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific, catalog number: 34096 )
  9. X-ray films: Amersham Hyperfilm ECL (GE Healthcare Life Sciences, catalog number: 28-9068-35 )
  10. Primary antibodies
    1. Rabbit anti-human Nav1.5 antibody (Alomone Labs, catalog number: ASC-013 )
    2. Mouse anti-Na+/K+ ATPase (Abcam, catalog number: ab7671 )
  11. Secondary antibodies
    1. Stabilized Goat Anti-Mouse IgG (H + L), Peroxidase Conjugated (10 μg/ml) (Thermo Fisher Scientific, catalog number: 32430 )
    2. Stabilized Goat Anti-Rabbit IgG (H + L), Peroxidase Conjugated (10 μg/ml) (Thermo Fisher Scientific, catalog number: 32460 )
  12. Protease inhibitors Cocktail (Roche Tablets, catalog number: 11 836 170 001 )
  13. DPBS with calcium and magnesium (DPBS+) (see Recipes)
  14. Lysis buffer LB1, LB2, LB3 (see Recipes)
  15. Phosphate-Buffered Saline (PBS) (see Recipes)
  16. Saline washing solution (SWS) (see Recipes)
  17. 3x Gel loading buffer (see Recipes)
  18. SDS-PAGE gel (see Recipes)
  19. 5x Running buffer (see Recipes)
  20. 10x Western blot transfer buffer (see Recipes)

Equipment

  1. 35 mm Dish (Thermo Fisher Scientific, catalog number: 153066 )
  2. 6-well cell culture plate (BD Biosciences, catalog number: 353046 )
  3. Cell scrapers (VWR International, catalog number: 734-2603 )
  4. 96-well Microtest Plate (Sarstedt, catalog number: 82.1581 )
  5. OrbitTM LS Low Speed Shaker (LABNET, catalog number: S-2030-LS )
  6. Rotating wheel (Noria R NR50, Ovan, catalog number: 10000-00062 )
  7. Microcentrifuge R5415 (Eppendorf, catalog number: 022621425 )
  8. Digital heat block (VWR International, catalog number: 460-3267 )
  9. Microplate Reader Benchmark plus (Bio-Rad Laboratories, catalog number: 170-6936 )
  10. Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )
  11. Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 170-3935 )
  12. PowerPac HC Power Supply (Bio-Rad Laboratories, catalog number: 164-5052 )

Software

  1. ImageJ software (available at http://rsb.info.nih.gov/ij ) (National Institute of Health, NIH)

Procedure

  1. Biotinylation
    Note: Perform all incubations on ice.
    1. Remove growth media from the cells that you want to study, cultured in 35 mm plates.
      Note: We used Human Embryonic Kidney HEK293 cells as experimental model. Cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 1% Penicilin-streptomycin and 1% Glumtamax at 37 °C and 5% CO2.
      We plated approximately 2.1 x 105 HEK293 cells in 35 mm dishes, but this value may vary depending on how fast your cell line grows (this will result in 70% of confluence 24 h later, which is the day of the transfection). Forty-eight hours after transfection we performed the Cell Surface Protein Biotinylation protocol.
    2. Wash cells twice with 1 ml of ice cold DPBS+.
    3. Incubate cells for 30 min on ice in the cold room with gentle rocking with 400 μl per plate of the biotin solution (2.5 mg/ml biotin reagent in DPBS+).
    4. Wash each plate for 5 min three times with 1 ml of cold 100 mM Glycine in DPBS+, on ice in the cold room with gentle rocking.
    5. Wash each plate for 5 min twice with 1 ml of cold 20 mM Glycine in DPBS+, as above.
    6. Lyse with 200 μl of lysis buffer LB3 into each plate and use cell scrapers to detach the cells. Collect the cell lysates in 1.5 ml tubes.
    7. For lysis, place the tubes in a rotating wheel at slow speed 1 h in the cold room.
    8. While the lysis is performed, prepare the Immobilized/ NeutrAvidin Ultralink beads: For each sample, take 40 μl of NeutrAvidin beads at 50% slurry and wash the beads twice with 0.5 ml DPBS+ and twice again with 0.5 ml LB2 (for washing, centrifuge 30 sec at 3,000 x g at 4 °C and remove the supernatant). After the last wash, resuspend the precipitated beads with 20 μl of LB3.
    9. Spin lysates at 16,000 x g (maximum speed) for 15 min at 4 °C.
    10. Transfer the supernatants (solubilized material) to 1.5 ml tubes. Keep 10-15% of each supernatant in another tube and store at -80 °C (these are the INPUT samples, and the rest of the supernatant will be referred to as PULL-DOWN samples).
    11. Incubate the pull-down samples with the 40 μl of NeutrAvidin beads prepared in step 8 overnight in the rotating wheel at slow speed in the cold room.
    12. Centrifuge the samples at 16,000 x g, 30 sec at 4 °C.
    13. Wash beads once with LB3, twice with LB2, twice with SWS and once with LB1. Each time, add 1 ml of the appropriate solution and centrifuge at 16,000 x g, 30 sec at 4 °C.
    14. Resuspend the precipitated proteins in 25 μl of 2x gel loading buffer.
    15. Heat the samples at 70 °C for 10 min in the heat block.
    16. Keep pull-down samples at -20 °C until SDS-PAGE and Western blot.

  2. Western blot
    1. Use part of the input samples to quantify the protein using the BCA Protein Assay Reagent alongside BSA standards, following the manufacturer's directions. From each input sample, transfer equal amounts of protein to a new tube and mix with 3x gel loading buffer. Heat the samples at 70 °C for 10 min.
      Note: Other protein quantification methods can be used and are equally efficient.

    2. Load in an SDS-PAGE gel the aliquots of the inputs prepared in step B-1 and the total volume of the pull-down samples (without the beads). Before loading, spin down the samples to pellet the beads.
    3. Run the gel at 40 V until samples have run through the stacking gel (30 min approximately). At that point, increase voltage to 100 V until the blue dye front has nearly run out of the gel.
    4. Prepare the sandwich: Soak the gel 15 min shaking in Western blot transfer buffer. Activate the Polyvinylidene difluoride (PVDF) membrane by soaking it for 10 sec with methanol, then twice with water and 10 min with Western blot Transfer Buffer. Also, wet two sheets of filter paper and two fiber pads with Western blot Transfer Buffer. Construct the sandwich with the gel on the cathode side and the membrane on the anode side, with a sheet of filter paper and a fiber pad at each side.
    5. Transfer the proteins to the PVDF membrane at 80 V for 2 h in the cold room.
    6. Remove the membrane from the sandwich and block it with 5% nonfat milk in PBS with 0.1% Tween-20 1 h at room temperature.
      Note: All Western blot incubations were performed in a shaker at low speed.
    7. Incubate the membrane with primary antibody overnight at 4 °C.
      Note: We used antibodies against human Nav1.5 (protein of interest; rabbit polyclonal, at 1:1,000 in 5% non-fat milk in PBS with 0.1% Tween-20) and Na+/K+ ATPase (membrane protein used as control, mouse, at 1:5,000 in 5% non-fat milk in PBS with 0.1% Tween-20).
    8. Wash three times with PBS with 0.1% Tween-20 to completely cover the membrane (approximately 20 ml), 10 min each, at room temperature.
    9. Incubate with the appropriate secondary antibody 1 h at room temperature.
      Note: We used peroxidase conjugated- goat anti-rabbit or anti-mouse IgG antibodies for Nav1.5 and Na+/K+ ATPase, respectively (at 1:2,000 in 5% non-fat milk-PBS with 0.1% Tween-20).
    10. Wash twice with PBS with 0.1% Tween-20 to completely cover the membrane (approximately 20 ml), 10 min each, at room temperature.
    11. For signal development, use the chemiluminescence substrate and follow the manufacturer's instructions. Remove excess reagent and cover the membrane in transparent plastic wrap.
    12. Detect the protein of interest by exposure to X-ray films (Figure 1).
      Note: For normalization purposes, a membrane protein should also be assessed as control.


      Figure 1. Western Blot detection of Nav1.5 and Na+/K+ ATPase proteins performed after cell surface biotinylation from WT and mutant cells

  3. Quantification
    1. Scan the films in high resolution using the transparency mode.
    2. Use the ImageJ software for band quantification. Follow the user's guide instructions to determine intensity values for each band as the integrated density (sum of pixel values) within a fixed area.
      Note: When the aim is to compare WT and mutant conditions within multiple replicates, proceed as follows:
      1. Determine intensity values for the bands corresponding to WT and mutant conditions for the membrane protein used as control (ICWT and ICMUT, respectively) and WT and mutant condition for the protein of interest (IWT and IMut, respectively).
      2. Calculate the ratio between ICWT and ICMUT to obtain the normalization factor (Nf):
        ICWT/ICMUT = Nf
      3. Multiply IMut by the normalization factor calculated in the previous step to obtain the corrected mutant condition intensity value (IC Mut):
        IMut x Nf = IC Mut
      4. Divide IC Mut by IWT to obtain the expression of the mutant condition relative to IWT.
        IC Mut/IWT = mutant relative expression
      5. Calculate the average of the mutant relative expression values for all the replicates and perform statistical analysis to assess possible differences with respect to the WT (Figure 2).


        Figure 2. Bar graph shows the average of the intensity values obtained for each condition

Recipes

     Note: All chemical reagents were obtained from Sigma-Aldrich unless stated otherwise.

  1. DPBS+ (For 2 L)
    0.2 g of CaCl2
    0.4 g KCl
    0.4 g KH2PO4
    0.2 g MgCl2.6H2O
    16 g NaCl
    4.32 g Na2HPO4.7H2O
    To avoid forming an insoluble precipitate, add everything except CaCl2, and have it stirring in the cold room close to correct volume. While stirring and when cold, add CaCl2, and continue stirring for at least 10 min until the solution becomes clear. Add milli-Q water to the final volume of 2 L.
  2. LB1
    50 mM Tris/HCl (pH 7.4)
    150 mM NaCl
    1 mM EDTA
  3. LB2
    LB1 plus 1% (w/v) Triton X-100
    To mix well, place the solution 1 h in cold room in the rotating wheel at slow speed.
  4. LB3
    For every 10 ml of solution LB2, add 1 tablet of protease inhibitors Cocktail right before use.
  5. 1x PBS
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Add mili-Q water for bring the final volume to 1 L.
  6. SWS
    0.1% Triton X-100 in PBS (pH 7.4)
    350 mM NaCl
    5 mM EDTA
  7. 3x gel loading buffer
    180 mM Tris/HCl (pH 6.8)
    7.5% SDS
    30% glycerol
    0.051% Bromophenol blue
    150 mM DTT
  8. SDS-PAGE gel (for 2 gels 1.5 mm thick)
    1. Stacking (4%)
      4.65 ml H2O
      1.88 ml 0.5 M Tris (pH 7.4)
      0.75 ml Acrylamide: Bisacrylamide 30% solution 37.5:1
      75 μl 10% SDS
      45 μl 10% Ammonium persulfate
      15 μl N, N, N', N'-tetramethylethylenediamine (TEMED)
    2. Resolving (4%)
      9.35 ml H2O
      3.75 ml 1.5 M Tris (pH 8.8)
      1.46 ml Acrylamide: 30% Bisacrylamide solution 37.5:1
      150 μl 10% SDS
      69 μl Ammonium persulfate 10%
      23.1 μl N, N, N', N'-tetramethylethylenediamine (TEMED)
  9. 5x Running buffer (For 1 L)
    5 g SDS
    144 g glycine
    30 g Tris
  10. 10x Western blot transfer buffer (For 1 L)
    10 g SDS
    24.24 g Tris
    111.75 g glycine
    Before use, make up 1x and add 20% Methanol.

Acknowledgments

The protocol was used in: Riuró et al. (2013); and Tarradas et al. (2013), but was adapted from a previously published paper: Cuartero et al. (2012). Funding sources at the time the protocol was developed included a grant from the Spanish Ministerio de Sanidad y Consumo to M. Verges (PI07/0895); Fellowship from the Príncipe Felipe Research Center (CIPF) to Y. Cuartero (PR 01/2007); and Ramón y Cajal contract from the Spanish Ministerio de Educación y Ciencia to M. Verges. Funding when it was implemented imcluded “La Caixa” Foundation to R. Brugada; Centro Nacional de Investigaciones Cardiovasculares (CNIC) Translational to R. Brugada (CNIC-03-2008); Ministerio de Sanidad y Consumo to R. Brugada (PI08/1800); Ministerio de Sanidad y Consumo fellowships or contracts (FI09/00336, CD09/00055, CD10/00275, CD11/00063 and PI2008/1800); Univ. of Girona fellowships to H. Riuró (BR2012/47); Ministerio de Sanidad y Consumo: Red Cooperativa de Insuficiencia Cardiaca (REDINSCOR) RD06/03/0018; and Sociedad Española de Cardiología (2011, Investigación Básica).

References

  1. Cuartero, Y., Mellado, M., Capell, A., Alvarez-Dolado, M. and Verges, M. (2012). Retromer regulates postendocytic sorting of beta-secretase in polarized Madin-Darby canine kidney cells. Traffic 13(10): 1393-1410.
  2. Riuró, H., Beltran‐Alvarez, P., Tarradas, A., Selga, E., Campuzano, O., Vergés, M., Pagans, S.,Iglesias, A., Brugada, J. and Brugada, P. (2013). A missense mutation in the sodium channel β2 subunit reveals SCN2B as a new candidate gene for brugada syndrome. Hum Mutat 34(7):961-966.
  3. Tarradas, A., Selga, E., Beltran-Alvarez, P., Perez-Serra, A., Riuro, H., Pico, F., Iglesias, A.,Campuzano, O., Castro-Urda, V., Fernandez-Lozano, I., Perez, G. J., Scornik, F. S. and Brugada, R.(2013). A novel missense mutation, I890T, in the pore region of cardiac sodium channel causes brugada syndrome. PLoS One 8(1): e53220. 

简介

特异性分离和定量细胞表面膜中的蛋白质的好方法是利用生物素化技术。 它包括在裂解细胞之前用生物素试剂标记细胞表面蛋白,并通过NeutrAvidin下拉分离这些标记的蛋白。 然后,将样品进行SDS-PAGE分离,转移至PVDF膜并用特异性抗体探测。 细胞表面表达的定量通过密度测量对应于感兴趣的蛋白质的条带并随后通过膜蛋白质(作为对照)标准化来完成。

关键字:细胞表面蛋白的生物素化, SDS-PAGE (同时修改英文?), 蛋白质印迹法, 膜蛋白

材料和试剂

  1. 人胚胎肾HEK293细胞(Health Protection Agency Culture Collections,目录号:96121229)
  2. 生物素试剂:EZ-link Sulfo-NHS-LC-LC-生物素(Thermo-Pierce,目录号:21338)
  3. 电泳级甘氨酸(Sigma-Aldrich,目录号:G8898)
  4. 固定化/NeutrAvidin Ultralink树脂(Thermo-Pierce,目录号:53150)
  5. Pierce BCA蛋白测定试剂盒(Thermo-Pierce,目录号:23227)
  6. 聚偏二氟乙烯(PVDF)膜(GE Healthcare Life Sciences,目录号:RPN303F)
  7. 脱脂奶粉(AppliChem GmbH,目录号:A0830)
  8. 化学发光底物:超级信号西飞最大灵敏度底物(Thermo Fisher Scientific,目录号:34096)
  9. X射线胶片:Amersham Hyperfilm ECL(GE Healthcare Life Sciences,目录号:28-9068-35)
  10. 一抗
    1. 兔抗人Na v 1.5抗体(Alomone Labs,目录号:ASC-013)
    2. 小鼠抗Na +/+/K + ATP酶(Abcam,目录号:ab7671)
  11. 二抗
    1. 稳定的山羊抗小鼠IgG(H + L),过氧化物酶缀合的(10μg/ml)(Thermo Fisher Scientific,目录号:32430)
    2. 稳定的山羊抗兔IgG(H + L),过氧化物酶缀合的(10μg/ml)(Thermo Fisher Scientific,目录号:32460)
  12. 蛋白酶抑制剂Cocktail(Roche Tablets,目录号:11 836 170 001)
  13. DPBS与钙和镁(DPBS + )(参见配方)
  14. 裂解缓冲液LB1,LB2,LB3(参见配方)
  15. 磷酸盐缓冲盐水(PBS)(参见配方)
  16. 盐水洗涤液(SWS)(见配方)
  17. 3x凝胶加样缓冲液(见配方)
  18. SDS-PAGE凝胶(参见配方)
  19. 5x运行缓冲区(参见配方)
  20. 10x Western印迹转移缓冲液(参见配方)

设备

  1. 35mm皿(Thermo Fisher Scientific,目录号:153066)
  2. 6孔细胞培养板(BD Biosciences,目录号:353046)
  3. 细胞刮刀(VWR International,目录号:734-2603)
  4. 96孔微量测试板(Sarstedt,目录号:82.1581)
  5. 轨道 TM LS低速振荡器(LABNET,目录号:S-2030-LS)
  6. 旋转轮(Noria R NR50,Ovan,目录号:10000-00062)
  7. 微量离心机R5415(Eppendorf,目录号:022621425)
  8. 数字热块(VWR International,目录号:460-3267)
  9. 微量板阅读器基准加(Bio-Rad Laboratories,目录号:170-6936)
  10. Mini-PROTEAN Tetra Cell(Bio-Rad Laboratories,目录号:165-8000)
  11. Mini Bio-Blot Module(Bio-Rad Laboratories,目录号:170-3935)
  12. PowerPac HC电源(Bio-Rad Laboratories,目录号:164-5052)

软件

  1. ImageJ软件(可在 http://rsb.info.nih.gov/ij )(国家 卫生研究所,NIH)

程序

  1. 生物素化
    注意:在冰上进行所有孵育。
    1. 从要研究的细胞中取出生长培养基,培养在35 mm平板上 注意:我们使用人胚胎肾HEK293细胞作为实验模型。将细胞在37℃和5%CO 2下维持在补充有10%胎牛血清,1%青霉素 - 链霉素和1%Glumtamax的Dulbecco's Modified Eagle's培养基中。
      我们在35mm皿中铺平大约2.1×10 5个HEK293细胞,但是该值可以根据细胞系生长的快慢而变化(这将导致24小时后70%的汇合,这是转染的天)。转染后48小时,我们进行细胞表面蛋白生物素化方案。
    2. 用1ml冰冷的DPBS + 洗涤细胞两次。
    3. 在冰室中在冰上温育细胞30分钟,同时用400μl每板生物素溶液(2.5mg/ml生物素试剂,在DPBS中)轻轻摇动。
    4. 在冰箱中,在冰箱中,在温和摇动的情况下,用1ml冷的100mM甘氨酸的DPBS溶液洗涤每个板5分钟,每次3分钟。
    5. 用如上所述的DPBS + 中的1ml冷的20mM甘氨酸洗涤每个板5分钟两次。
    6. 用200μl裂解缓冲液LB3裂解到每个板中,并使用细胞刮刀分离细胞。收集细胞裂解物在1.5毫升管
    7. 对于裂解,将管在冷室中以慢速1小时置于旋转轮中
    8. 在进行裂解时,制备固定化/NeutrAvidin Ultralink珠:对于每个样品,取40μl50%浆液的NeutrAvidin珠,并用0.5ml DPBS +洗涤珠两次,再用0.5μl ml LB2(用于洗涤,在4℃以3,000xg离心30秒,并除去上清液)。最后一次洗涤后,用20μlLB3重悬沉淀的珠子。
    9. 在4℃下以16,000×g(最大速度)旋转裂解物15分钟。
    10. 将上清液(溶解的材料)转移到1.5ml管。 将每种上清液的10-15%保存在另一个管中,并储存在-80℃(这些是INPUT样品,其余的上清液将称为PULL-DOWN样品)。
    11. 将下拉样品与步骤8中制备的40μlNeutrAvidin珠子一起在旋转轮中以低速在冷室中孵育过夜。
    12. 以16,000×g离心样品,在4℃下30秒
    13. 用LB3洗涤珠一次,用LB2洗两次,用SWS洗两次,用LB1洗一次。 每次,加入1ml适当的溶液,并在16,000×g离心,在4℃下30秒。
    14. 重悬沉淀的蛋白质在25微升2×凝胶加载缓冲液。
    15. 在加热块中将样品在70℃加热10分钟
    16. 将下拉样品保持在-20°C,直到SDS-PAGE和Western印迹
  2. 蛋白质印迹
    1. 使用部分输入样品,使用BCA蛋白测定试剂以及BSA标准品,按照制造商的说明书来定量蛋白质。从每个输入样品,将等量的蛋白质转移到新管中,并与3x凝胶上样缓冲液混合。将样品在70℃加热10分钟。
      注意:可以使用其他蛋白质定量方法,并且同样有效。
    2. 在SDS-PAGE凝胶中加载步骤B-1中制备的输入的等分试样和下拉样品(无珠)的总体积。在装载之前,旋转样品以使珠粒沉淀
    3. 在40 V运行凝胶,直到样品已经通过堆叠凝胶(大约30分钟)。此时,将电压提高到100 V,直到蓝色染料前沿几乎用完凝胶
    4. 准备三明治:浸泡凝胶15分钟在Western印迹转移缓冲液摇动。通过用甲醇浸泡10秒,然后用水漂洗两次并用Western印迹转移缓冲液10分钟来激活聚偏二氟乙烯(PVDF)膜。此外,湿两张滤纸和两个纤维垫与Western印迹转移缓冲液。用阴极侧的凝胶和阳极侧的膜构建三明治,在每侧有一张滤纸和一个纤维垫。
    5. 转移蛋白质在PVDF膜在80伏在寒冷的房间2小时
    6. 从夹心中取出膜,用含有0.1%Tween-20的PBS中的5%脱脂牛奶在室温下封闭1小时。
      注意:所有的蛋白质印迹孵育在振荡器中以低速进行。
    7. 孵育膜与一抗在4℃过夜。
      注意:我们使用针对人Na v 1.5(感兴趣蛋白;兔多克隆,在含0.1%Tween-20的PBS中的5%脱脂奶中以1:1,000的比率)的抗体和Na (在具有0.1%Tween-20的PBS中的5%脱脂奶中以1:5,000的比率使用作为对照的膜蛋白)/em>
    8. 用含有0.1%Tween-20的PBS洗涤三次,以在室温下完全覆盖膜(约20ml),每次10分钟。
    9. 在室温下与适当的二抗孵育1小时 注意:我们使用过氧化物酶缀合的山羊抗兔或抗小鼠IgG抗体用于Na + 1.5和Na + +/+/+ - + > ATP酶(在含有0.1%Tween-20的5%脱脂牛奶-PBS中的1:2,000)。
    10. 用含有0.1%Tween-20的PBS洗涤两次,以在室温下完全覆盖膜(约20ml),每次10分钟。
    11. 对于信号发展,使用化学发光底物,并按照制造商的说明。取出多余的试剂,并用透明塑料包装膜覆盖膜
    12. 通过暴露于X射线胶片检测感兴趣的蛋白质(图1)。
      注意:为了标准化目的,膜蛋白也应作为对照。


      图1. Na v 1.5和Na + /K + ATP酶蛋白在WT和突变细胞的细胞表面生物素化后进行 />
  3. 定量
    1. 使用透明模式以高分辨率扫描胶片。
    2. 使用ImageJ软件进行带定量。 按照用户指南说明,确定每个波段的强度值,作为固定区域内的积分密度(像素值之和)。
      注意:当目的是在多个重复中比较WT和突变体条件时,按以下步骤进行:
      1. 确定对应于用作对照的膜蛋白(分别为IC 和IC MUT )的WT和突变条件的条带的强度值,以及蛋白的WT和突变条件 (分别是I sub WT和I sub Mut)。
      2. 计算IC sub WT和IC sub MUT之间的比率以获得归一化因子(N sub):< br />
        IC WT /IC MUT = N f
      3. 通过在前一步骤中计算的归一化因子乘以I mut以获得校正的突变条件强度值(IC sub):
        I Mut x N f = I C Mut
      4. 通过I sub WT分离IC sub以获得相对于I sub WT的突变条件的表达。
        IC /I WT =突变体相对表达
      5. 计算所有重复的突变体相对表达值的平均值,并进行统计分析以评估相对于WT的可能差异(图2)。


        图2.条形图显示了每个条件获得的强度值的平均值

食谱

     注:除非另有说明,所有化学试剂均获自Sigma-Aldrich。

  1. DPBS + (对于2 L)
    0.2g CaCl 2·h/v 0.4克KCl
    0.4g KH 2 PO 4 sub/
    0.2g MgCl 2 6H 2 O
    16克NaCl
    4.32g Na 2 HPO 4 sub 。 7H O
    为了避免形成不溶性沉淀,添加除了CaCl 2 2以外的所有物质,并在冷室中在接近正确体积的情况下搅拌。 在搅拌和冷却时,加入CaCl 2,并继续搅拌至少10分钟,直到溶液变澄清。 加入milli-Q水至最终体积为2L。
  2. LB1
    50mM Tris/HCl(pH7.4) 150mM NaCl 1mM EDTA
  3. LB2
    LB1加1%(w/v)Triton X-100 为了充分混合,将溶液在旋转轮中的低温室中缓慢地放置1小时
  4. LB2
    对于每10ml溶液LB2,在使用前加入1片蛋白酶抑制剂混合物
  5. 1x PBS
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
  6. SWS
    0.1%Triton X-100的PBS(pH7.4)中 350 mM NaCl 5 mM EDTA
  7. 3x凝胶上样缓冲液
    180mM Tris/HCl(pH6.8)
    7.5%SDS
    30%甘油 0.051%溴酚蓝
    150 mM DTT
  8. SDS-PAGE凝胶(用于2个凝胶,1.5mm厚)
    1. 堆叠(4%)
      4.65ml H 2 O x / 1.88ml 0.5M Tris(pH7.4)
      0.75毫升丙烯酰胺:双丙烯酰胺30%溶液37.5:1
      75μl10%SDS
      45μl10%过硫酸铵
      15μlN,N,N',N'-四甲基乙二胺(TEMED)
    2. 解决(4%)
      9.35ml H 2 O x / 3.75ml 1.5M Tris(pH8.8)
      1.46ml丙烯酰胺:30%双丙烯酰胺溶液37.5:1 150μl10%SDS
      69μl过硫酸铵10%
      23.1μlN,N,N',N'-四甲基乙二胺(TEMED)
  9. 5x运行缓冲液(1升)
    5克SDS
    144 g甘氨酸
    30克Tris
  10. 10x Western印迹转移缓冲液(对于1L)
    10克SDS
    24.24克Tris
    111.75克甘氨酸 使用前,补充1x,加入20%甲醇

致谢

该方案用于:Riuró等人(2013);和Tarradas等人(2013),但改编自以前发表的论文:Cuartero等人(2012)。在议定书制定时的资金来源包括西班牙部长萨尼迪达和Consumo向M. Verges的赠款(PI07/0895);从PríncipeFelipe研究中心(CIPF)到Y. Cuartero(PR 01/2007)的奖学金;和Ramóny Cajal合同,从西班牙教育部长到西班牙部长M. Verges。资金实施时包括"La Caixa"基金会到R. Brugada; Centro Nacional de Investigaciones Cardiovasculares(CNIC)Translational to R. Brugada(CNIC-03-2008); Ministry of De Sanidad y Consumo to R. Brugada(PI08/1800);财政部长奖学金或合同(FI09/00336,CD09/00055,CD10/00275,CD11/00063和PI2008/1800);大学。的赫罗纳奖学金给H.Riuró(BR2012/47); Ministeria de Sanidad y Consumo:Red Cooperativa de Insuficiencia Cardiaca(REDINSCOR)RD06/03/0018;和SociedadEspañoladeCardiología(2011,InvestigaciónBásica)。

参考文献

  1. Cuartero,Y.,Mellado,M.,Capell,A.,Alvarez-Dolado,M.and Verges,M。(2012)。 Retromer调节极性Madin-Darby犬肾细胞中β-分泌酶的后内吞分选。 交通 13(10):1393-1410
  2. Riuró,H.,Beltran-Alvarez,P.,Tarradas,A.,Selga,E.,Campuzano,O.,Vergés,M.,Pagans,S.,Iglesias,A.,Brugada,J.and Brugada,P 。(2013)。 钠通道β2亚基的错义突变显示SCN2B 为新的brumada综合征的候选基因。 Hum Mutat 34(7):961-966。
  3. Tarradas,A.,Selga,E.,Beltran-Alvarez,P.,Perez-Serra,A.,Riuro,H.,Pico,F.,Iglesias,A.,Campuzano,O.,Castro-Urda, ,Fernandez-Lozano,I.,Perez,GJ,Scornik,FS和Brugada,R。(2013)。 在心脏钠通道的孔区域中的新型错义突变I890T引起brugada综合征。 a> PLoS One 8(1):e53220。 
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引用:Tarradas, A., Selga, E., Riuró, H., Scornik, F., Brugada, R. and Vergés, M. (2013). Cell Surface Protein Biotinylation and Analysis. Bio-protocol 3(16): e857. DOI: 10.21769/BioProtoc.857.
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