酶谱法检测功能的基质金属蛋白酶

Biology
 

Summary

本协议描述了一个用于检测培养上清或体液基质金属蛋白酶的活动为基础的检测。

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Hu, X., Beeton, C. Detection of Functional Matrix Metalloproteinases by Zymography. J. Vis. Exp. (45), e2445, doi:10.3791/2445 (2010).

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Abstract

基质金属蛋白酶(MMPs)是含有锌endopeptidases。他们降解蛋白质肽键的断裂。超过20基质金属蛋白酶已经确定,并分隔成六个小组,根据它们的结构和底物特异性(胶原酶,明胶酶,膜型[MT - MMP,stromelysins,matrilysins,和其他人)。基质金属蛋白酶在细胞的浸润,软骨退化,组织重构,伤口愈合和胚胎发育中发挥关键作用。因此,他们在正常的进程,并在许多疾病,如类风湿关节炎,癌症,慢性阻塞性肺疾病 1-6发病的参与。在这里,我们将重点对MMP - 2(明胶酶A,IV型胶原酶),基质金属蛋白酶广泛表达。我们将演示如何检测细胞培养上清液中MMP - 2的酶谱法,一种常用的,简单,但非常敏感的技术,在1980年首次描述C. Heussen EB Dowdle 7-10。这种技术是半定量的,因此它可以被用于确定测试样品基质金属蛋白酶的重组MMP的已知浓度的水平时,装载同一凝胶11。

聚丙烯酰胺凝胶含有十二烷基硫酸钠(SDS,线性的蛋白质)和明胶(MMP - 2的基板)装上含有基质金属蛋白酶(如细胞培养上清液,尿液或血清)的解决方案。样本缓冲区的目的是为了增加样品粘度(以方便装载凝胶),提供追踪染料(溴酚蓝;监测样本迁移),提供变性分子(线性蛋白质),并控制样品的pH值。蛋白质,然后允许电流下的迁移,在设计,以提供一个恒定的迁移率一个运行缓冲。迁移距离呈负相关的蛋白质的分子量(小分子蛋白通过凝胶中移动速度更快的比大的蛋白质,因此进一步下降凝胶迁移)。迁移后,凝胶放置在复性缓冲液使蛋白质,以重新获得接受高等教育的结构,酶的活性所必需的。凝胶,然后放置在一个发展中的缓冲区设计,允许蛋白酶消化其底。发展中的缓冲区也包含P - aminophenylmercuric醋酸(APMA)变为主动的基质金属蛋白酶激活非蛋白水解的基质金属蛋白酶。下一步,包括染色基板(在我们的例子明胶)。洗去多余的染料凝胶后,蛋白酶消化领域出现清晰的条带。乐队的更清晰,更集中的蛋白酶它包含。带染色强度可以通过光密度确定,如ImageJ软件使用,允许样品比较。

Protocol

1。加载和运行的凝胶

  1. 所有样品必须充分准备,保持酶的功能,采集后立即使用,或冷冻保存于-80 ° C。样品必须不包含还原剂(这样的β-巯基乙醇)或凝胶装载前煮沸。
  2. 打开袋录像带里面含有一种凝胶,录像带用去离子水冲洗。
    • 取出录像带的底部,并从凝胶顶部梳保护胶带。
    • 运行缓冲液(去离子水稀释至1X)冲洗井的三倍。
    • 凝胶放入微型电池,确保在录像带较小的侧面向内。凝胶张力楔锁定到位。迷你细胞允许并行运行一个或两个凝胶。
    • 填写与1X井水平以上运行缓冲区的顶部(内)室,检查有无渗漏。在泄漏的情况下,删除的缓冲区,并重新定位凝胶。
    • 下腔填充1X运行缓冲。
  3. 装入一个孔10μL蛋白质的分子标记。
    • 等量混合成井使用凝胶加载提示(每个样品之​​间的改变)的凝胶,凝胶样缓冲液和样品和负载。这些井可装载至20μL总。
  4. 迷你细胞上的盖子和电极线连接到电源。开关电源,并设置它运行在125 V恒定为90分钟。检查下腔线的小气泡的形成,表明当前的流通。
  5. 当运行您的第一个凝胶,迁移每15分钟监测方面取得的进展,使用溴酚蓝上样缓冲液作为一项指标包括。让凝胶运行,直到指示剂到达凝胶底部。

2。复性和发展的凝胶

  1. 对于每一个凝胶,准备1X复性缓冲和变性缓冲液200毫升,100毫升,无论是在去离子水。
  2. 当溴酚蓝跟踪染料到达凝胶底部,接通电源关闭,打开迷你细胞,取出凝胶。双方在录像带使用的胶刀(或称重锅铲)分开。切一个角落,以纪念凝胶的方向。
  3. 小心取出凝胶放入容器纸盒和地方与100毫升的复性缓冲。孵育30分钟,在室温下轻轻摇动。
  4. 删除复性缓冲,加入100毫升凝胶发展缓冲区。孵育30分钟,在室温下轻轻摇动。
  5. 删除发展中国家的缓冲区,并添加100毫升的发展缓冲的凝胶。孵育过夜(16-18小时),在37 ° C。
  6. 删除发展中国家缓冲区,并在室温下轻轻摇动去离子水冲洗三次(每次5分钟)。
  7. 扫描保存的蛋白质标准频段的精确定位,因为他们将成为少用或不可见的凝胶染色后的凝胶。
  8. 染色,加入20毫升SimplyBlue Safestain的凝胶,凝胶。轻轻摇动的温度在室温下孵育1小时。
  9. 卸下SimplyBlue SafeStain和色斑的100毫升或更多的去离子水凝胶在室温下轻轻摇动下一个小时。
  10. 为了获得更好的效果,更换新鲜去离子水,并培育,再在室温下轻轻摇动下的小时或更长时间。

3。数据分析

  1. 小心取出凝胶,水和一张塑料布保护。
  2. 扫描分辨率300 dpi或更高的凝胶。保存在TIFF格式的图像(图1A)。
  3. ImageJ(或其他类似的软件)测量的波段强度。
    • 打开ImageJ(图1A)TIFF文件。
    • 在黑色和白色的直观选择“图像>类型> 8位”(图1B)。
    • 使用矩形选择工具,勾勒出的第一个乐队,至少两次高于宽(这是一个软件的要求)绘制一个矩形。
    • 按“1”或选择“分析>凝胶>选择”第一线“乐队将概述。
    • 将出现一个新的矩形,将其移动到下一个波段,选择“分析>凝胶>选择下一个车道”。重复,直到所有频段选择(图1B)。
    • 按“3”或选择“分析>凝胶>剧情通道”,生成的每个波段的个人资料(图1C)情节。
    • 使用直线选择(应该已经自动选择)绘制基准线,使利益的高峰,是一个完全封闭的区域。
    • 选择棒工具,并在每个高峰,单击以选中它。
    • 选择“分析>凝胶>标签峰”,以获得与每个选定的高峰领域的一个表。这些数据可以绘制成这样(Figure 1D)或可归到一个频带的价值。这正常化池几个复制凝胶的值,以确定结果的统计学意义时,显得尤为重要。

4。代表性的成果

我们展示一个与不同的细胞培养上清中含有不同量的MMP - 2(图1A)的装入11井的凝胶。这种凝胶的直接观察表明在一些井之间的MMP - 2浓度明显的差异。例如,很显然,井#4和5包含得多好#11,甚至10号MMP - 2。客观量化的乐队,我们​​已经用ImageJ软件(图1B - D)密度测量,确认以及#11和水井#4和第5和一个约2倍的差异,在差异中MMP - 2的金额约4倍MMP - 2以及10号和水井#4和5之间的数额。

图1
图1。明胶酶谱法检测MMP - 2。一,扫描图像明胶凝胶。井数字表明在凝胶的顶部。 B,同在一个凝胶显示在黑色和白色为光密度。每个频带被选定在ImageJ的矩形。 A和B,光密度剖面的两个例子所示凝胶(带#4和#11)。直线绘制在每个峰的基地,创建一个封闭的区域。研发,绘制峰面积为A和B前(左)后(右)正常化带#1的密度所示的凝胶。

图2
图2这个数字演示如何酶谱可以作为一种半定量技术。我们已经加载在连续7口井连续稀释(按1:1稀释的步骤)和两个亲的MMP - 2和MMP - 2的带密度测量。

Discussion

我们已经演示了如何执行zymographic分析细胞培养上清中MMP - 2的。

加载凝胶上的样品量必须根据凭经验确定的起源和MMP利益。负荷是否过小会防止检测,同时装载过多可能会导致饱和,因为只有这么多基板一种蛋白酶可以消化在乐队的领域。如果有必要,样品可以在去离子水稀释前与样缓冲液,或开发时间混合,可以减少从隔夜到4个小时。它也有可能集中在一个解决方案使用集中器(如Millipore公司目录#UFC803024)的蛋白质。如果乐队仍隐约可见,它可能是必要制定一个较长时间的凝胶,甚至长达48小时。准备从组织培养上清样品时,应当指出,胎牛血清(和其他血清)中包含的蛋白酶可以影响的结果。出于这个原因,我们收集无血清培养基培养后,我们所有的上清。

在第2.9和2.10的协议中所描述的清洗需要删除的背景染色消化乐队。洗更长的时间将删除更多的背景,但也将暗淡的基材整个凝胶染色。如果需要更长的洗涤时间是必要的,我们建议扫描凝胶每隔几个小时,选择最好的对比度扫描。

这种技术可用于检测其他的蛋白酶。例如,可以检测到MMP - 9明胶凝胶体,也程度较低的MMP - 1,MMP - 8和MMP - 13,明胶是不是他们的首选基材。 MMP - 1和MMP - 13是最好的胶原蛋白的酶谱检测,而酪蛋白是首选基板和MMP - 11也可以检测MMP - 1,MMP - 3,MMP - 7,MMP - 12和MMP - 13 9。 MMP - 7(基质溶解)和胶原酶(MMP - 1和MMP - 13)是很难检测时,在低的水平目前在酪蛋白或明胶凝胶。此外肝素样品在凝胶加载时间显着提高MMP - 7的检测限。 MMP - 1和MMP - 13,肝素需要被添加到凝胶电泳运行后已经正在进行12。

由于酶谱措施酶的活性酶的变性和复性后,它会测量所有样品中的基质金属蛋白酶的活动。这包括酶,亲酶,并绑定到内源性抑制剂的酶(如MMP - 2)。不过,可以通过比较带密度的活性酶和酶,这将有一个稍高的分子量来确定样品中的基质金属蛋白酶激活水平。

酶谱法往往是不足够的识别MMP。 MMP与已知分子量标准的迁移水平的比较,帮助识别,但应该指出,这些标准包含还原剂和非还原条件下使用时,他们可能会表示不同分子量9。一个选项是一种可溶性的重组MMP的负载测试样品相同凝胶。然而,与其他蛋白质基质金属蛋白酶可能诱导表观分子量的变化。选择性MMP抑制剂可以被添加到发展缓冲区作为药理工具孵化期间的凝胶(或部分削减了一半的凝胶)的身份interest.A第二个方法(免疫印迹,免疫细胞化学法或ELISA)MMP建议帮助鉴定MMP利益。应该指出,Western印迹检测限制通常比zymographic凝胶低得多,可能会导致假阴性结果使用这种技术时。

Disclosures

没有利益冲突的声明。

Acknowledgements

我们要感谢S.雷斯勒(博士,贝勒医学院分子与细胞生物学部)建议使用的蛋白质集中在细胞培养上清液中基质金属蛋白酶的浓度增加。

这个项目是由夫人祈福长老白格雷厄姆赋研究基金和美国国立卫生研究院/ NIAMS(AR059838)对CB的赠款支持。内容完全是作者的责任,并不一定代表美国国立卫生研究院的官方意见。

Materials

Name Company Catalog Number Comments
Xcell SureLock Mini-Cell CE mark electrophoresis apparatus Invitrogen EI0001
Power supply (model 302) VWR international 93000-744
Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells Invitrogen EC61752BOX
Blue Juice gel loading buffer Invitrogen 10816015
Gel loading tips VWR international 53509-015
Protein molecular weight standard Invitrogen LC5800
Novex Tris-Glycine-SDS running buffer Invitrogen LC2675
Novex zymogram renaturing buffer Invitrogen LC2670
Novex zymogram developing buffer Invitrogen LC2671
SimplyBlue SafeStain Invitrogen LC6060
Epson Perfection 4490 Photo Scanner Amazon n/a
ImageJ software http://rsbweb.nih.gov/ij/ n/a Authored by W. Rasband, NIH/NIMH

DOWNLOAD MATERIALS LIST

References

  1. Luo, J. The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex. Cerebellum. 4, 239-245 (2005).
  2. Pasternak, B., Aspenberg, P. Metalloproteinases and their inhibitors - diagnostic and therapeutic opportunities in orthopedics. Acta Orthop. 80, 693-703 (2009).
  3. Kessenbrock, K., Plaks, V., Werb, Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 141, 52-67 (2010).
  4. Lagente, V., Boichot, E. Role of matrix metallopreoteinases in the inflammatory process of respiratory diseases. J. Mol. Cell. Cardiol. 48, 440-444 (2010).
  5. Rodríguez, D., Morrison, C. J., Overall, C. M. Matrix metallopreoteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics. Biochim. Biophys. Acta. 1803, 39-54 (2010).
  6. Bourboulia, D., Stetler-Stevenson, W. G. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion. Semin. Cancer Biol. Forthcoming (2010).
  7. Heussen, C., Dowdle, E. B. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Analytical Biochem. 102-196 (1980).
  8. Lombard, C., Saulnier, J., Wallach, J. Assays of matrix metalloproteinases (MMPs) activities: a review. Biochimie. 87, 265-272 (2005).
  9. Snoek-van Beurden, P. A., Von den Hoff, J. W. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques. 38, 73-83 (2005).
  10. Kupai, K., Szucs, G., Cseh, S., Hajdu, I., Csonka, C., Csont, T., Ferdinandy, P. Matrix metalloproteinase activity assays: importance of zymography. J. Pharmacol. Toxicol. Methods. 61, 205-209 (2010).
  11. Kleiner, D. E., Stetler-Stevenson, W. G. Quantitative zymography: detection of pictogram quantities of gelatinases. Anal. Biochem. 218, 325-329 (1994).
  12. Yu, W. H., Woessner, J. F. Jr Heparin-enhanced zymographic detection of matrilysin and collagenases. Anal. Biochem. 293, 38-42 (2001).

Comments

17 Comments

  1. Please suggest me the procedure for the extraction method of Tumors tissues from Rats and Canine for Gelatin zymography for MMPs

    Reply
    Posted by: Anonymous
    December 27, 2010 - 6:32 AM
  2. Hello, I am a young Algerian scholar and I would know if you use a single separating gel from what I saw in your video. Since me in my gel there are two parts: Gel concentration gel separation
    I also know the program or software you used to measure the activity of MMPs
    Thanks for your answer

    Reply
    Posted by: Anonymous
    December 20, 2011 - 3:59 PM
  3. When you have two bands close together, as in your gel with pro-MMP² and MMP² bands, do you do the same quantification procedure? How dŒs image j know which band it is quantitating if the box you have drawn contains both bands?

    Reply
    Posted by: Helicia P.
    May 31, 2012 - 5:31 PM
  4. In most cases you can draw two boxes, one above the other, if you want to quantify them separately. If they are too close, try running the gel longer for better separation.

    Reply
    Posted by: Christine B.
    November 6, 2012 - 6:26 PM
  5. Thank you for this very detailed procedure! It was very helpful. I am analyzing MMP (unknown type) in synovial fluid from human patients. When you performed teh above experiment did you do a Lowry BSA assay before?

    Reply
    Posted by: Shaili S.
    August 2, 2012 - 11:46 AM
  6. We did not but we were using culture supernatants. In the case of synovial fluid I think dowing a Lowry before hand is a good idea.

    Reply
    Posted by: Christine B.
    November 6, 2012 - 6:25 PM
  7. Good morning, i thank you for this exelent video, but if i can ask you one question, it' for how can i applicate the data analysis for gel zymographique i have not logiciel for thid . thank you

    Reply
    Posted by: anissa m.
    November 6, 2012 - 6:20 PM
  8. This software is freely downloadable from the NIH website listed in the materials.

    Reply
    Posted by: Christine B.
    November 6, 2012 - 6:24 PM
  9. Hello, thank you for the video, it is really helpful. I am interested in MMP-9, but the concentration in the medium is low. I am considering of using a concentrator, but I don't know what size i need to look for, different membranes come in different sizes, what would be the best for detecting both MMP-² and -9?

    Reply
    Posted by: Jenny K.
    November 14, 2012 - 7:19 AM
  10. Hi Jenny, we are glad our video-article can help you in your assays. To concentrate your MMPs, you need a concentrator that will concentrate proteins in the molecular weight range of your MMPs of interest. MMP-² has a MW of 63 kDa (7² kDa in the pro form) and MMP-9 has a MW of 64-67 kDa (9² kDa in the pro form). Millipore sells concentrators (Amicon) for proteins of different sizes. I suggest you use those for proteins of 50 kDa or more. Hope this helps!

    Reply
    Posted by: Anonymous
    March 14, 2013 - 6:59 PM
  11. Dear Sir,
    I am analyzing MMP-2 and MMP-9 in human serum by zymography method. Can I use serum and load in the gel?? and to use Recombinant Human MMP-9 as an standard... Do you think by using serum i can identify and get a band for MMP-2 and 9

    Reply
    Posted by: Yousef C.
    January 13, 2014 - 5:58 AM
  12. Yes, you can definitely detect MMPs in serum using this technique.

    Reply
    Posted by: Christine B.
    March 30, 2014 - 9:43 PM
  13. Hello,

    Did it ever happen to you that the ladder separated nicely until a point, and then migrates together as a band? Do you have any idea what could be the case?

    Thank you very much in advance.

    Reply
    Posted by: Anca R.
    February 24, 2014 - 10:31 AM
  14. This is not a problem we have had. The only thing I can think of is that the protein ladder doesn't match the gel density. Or did you not load enough of the ladder? In that case it is possible that what you see at the end is only the migration marker and that the bands of proteins are too faint to see.

    Reply
    Posted by: Christine B.
    March 30, 2014 - 9:42 PM
  15. any other scanner can be used for this??? Please reply me as soon as possible.....

    Reply
    Posted by: Yousef C.
    February 25, 2014 - 12:58 AM
  16. Any scanner that is setup to scan transparencies and gels can be used.

    Reply
    Posted by: Christine B.
    March 30, 2014 - 9:40 PM
  17. hai,

    i am working with mmp2 and mmp9 in chicken sperm samples particularly in plasma , we are not getting cleaved bands in gelatin zymography, its looking like SDS PAGE band , kindly suggest me how to improve my work .am using 4% gelatin in 8% resolving gel . i tried 19 and 36 hours of incubation time in developing buffer .

    Reply
    Posted by: Vinoth A.
    July 30, 2014 - 11:08 AM

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