The authors are grateful for the support and advice of Nikolaus Grigorieff. We thank Anna Loveland, Axel Brilot, Chen Xu, and Mike Rigney for helpful discussions and EM guidance. This work was funded by the National Science Foundation, Award No. 1157892. The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.

注意：下面的协议被设计为一个复杂的纯化从4升细胞培养物，细胞的大约40克湿重。一旦制备，所有缓冲区应储存在4℃并且在一个月它们的制备中使用。还原剂和蛋白酶抑制剂仅加入缓冲液在使用前。 1.全细胞提取的制备串联亲和纯化<ol><li> S.生长酵母细胞 <ol><li>连胜所需的TAP标记S.酵母菌株从存储器（-80°C）到酵母蛋白胨葡萄糖（YPD）板。孵育3 - 5天（3...

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该TAP方法利用两个标签来平衡需要严格和选择性的结合到亲和树脂保持接近生理溶液条件的愿望。这种平衡的作用是保持与净化后的特征相互作用的因子（S）标签蛋白的稳定的相互作用（S）。此外，个别的TAP标记的ORFs可从商业来源，这样就可以得到具有为任何酵母复杂的标签化蛋白质的任何酵母菌株。保留的复合物的完整性和资源，以测试在用于纯化的复杂使用不同标记的蛋白质亚基的可用性?...

许多主要的细胞过程是由大的蛋白质和蛋白质-RNA复合物1进行。一个显著瓶颈进行这种复合物的生物物理和结构研究是获得并在适当浓度的合适的质量（ 即 ，均匀性）它们。从天然来源隔离一个复杂的具有许多优点，包括护亚单位的相关转录后和/或翻译后修饰和投保适当的复杂的装配。然而，大蜂窝络合物以低拷贝数通常存在于细胞和纯化必须高效和近生理条件下发生，以确保复杂的完整性。来自真核来源纯化络合物是特别具有挑战性的，并且可以在经济上令人望而却步。因此，是有效率和产生均匀复杂的策略或方法是高度期望的。 这种策略已经成功地从净化真核细胞的天然复合物为他们的初步定性是串联亲和纯化（TAP）的方法2,3。该方法TAP最初设计在复杂的纯化由芽殖酵母（ 酿酒酵母）天然蛋白与之交互因子（S）2。该方法TAP利用了两个标签，串联融合到同一个蛋白质编码基因序列的每个标签。被选择的代码，以便平衡需要紧和选择性结合的亲和树脂，以保持接近生理溶液条件的愿望。这种平衡的作用是保持与净化后的特征相互作用的因子（S）标签蛋白的稳定的相互作用（S）。的基因组引入的TAP标记被放置在末端（C-末端）的蛋白质编码基因的和由一个序列编码的钙调蛋白结合肽（CBP），随后通过蛋白A的 - 加成刚刚超过20千道尔顿到标签蛋白。 CBP短，26个氨基酸，并通过识别的〜17 kDa蛋白钙调素在钙的存在下用K 的D 10 -9量级M 4上。蛋白A标记是较大，由58残两个重复与重复之间的短连接体。每58个氨基酸重复由免疫球蛋白G（IgG）的用K D〜10 -8 M 5识别。这两个标记之间成立的TEV蛋白酶，从烟草蚀纹病毒6,7内肽酶的识别位点。 如图1所示，在TAP标记蛋白的方法的第1亲和性步骤经由蛋白A的标记的蛋白质是由在加入TEV蛋白酶，位点特异性之间切割的洗脱上柱裂解绑定至IgG树脂这两个标签。这是一个必要的步骤为IgG的互动和蛋白A是非常强的，只能解变性的条件下得到充分的扰动。由于缺乏公关otein一个标记，该蛋白质结合到CaM的树脂在钙的存在下，并从该树脂与另外的金属离子螯合剂EGTA（乙二醇四乙酸）（ 图1）的洗脱。 引进TAP方法，它在一个大规模研究用于生成不久后，“地图”在S.复杂的相互作用酵母 8。重要的是，作为这种努力的整个酵母-TAP标记开放阅读框（ORF）库的结果，以及个别的TAP标记的ORF 9是可从商业来源。因此，可以得到具有为任何酵母复杂的标签化蛋白质的任何酵母菌株。在TAP方法还刺激了修改或在TAP标记的变体，其中包括：其用于从其它真核以及细菌细胞10,11配合物的纯化使用;一个“拆分标签”，其中蛋白质A和CBP被放置在不同的蛋白质12的设计;和TAGS改变，以便容纳研究者的需要，如复合物的 Ca 2+或EGTA 13的灵敏度。 在这两个仪器和方法学的最新进展已经导致了结构确定在电子显微镜（EM）的应用显著的进步，已导致高，接近大分子复合14的原子分辨率的图像。通过获得EM复杂的分辨率，但是，仍然在研究中的复杂的质量而定。这项研究利用了TAP标签的方式从S.净化酵母 U1的核蛋白，18亚基（〜0.8 MDA）低拷贝数核糖核蛋白复合物，是剪接体15,16的一部分。一些已采取步骤以纯化这种复合物，例如，它是均匀的和适当的浓度。在纯化的各阶段中遇到的潜在问题被描述，并采取克服CHALL策略恩格斯强调。通过仔细评估和净化优化步骤中，U1核蛋白纯化是适用于负染色和电子显微镜冷冻（冷冻电镜）研究数量质量和。用于结构研究天然复合物的纯化的优化TAP方法协议在本文中描述。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>S. cerevisiae TAP tagged strain</td>
			<td>Open Biosystems</td>
			<td>YSC1177</td>
			<td>This is the primary yeast strain used to develop the TAP protocol. Its background is S288C: ATCC 201388: MATa, his3&#916;1, leu2&#916;0, met15&#916;0, ura3&#916;0, SNU71::TAP::HIS3MX6</td>
		</tr>
		<tr>
			<td>Coffee grinder</td>
			<td>Mr. Coffee</td>
			<td>IDS77</td>
			<td>Used for cell lysis</td>
		</tr>
		<tr>
			<td>Hemocytometer, Bright Line</td>
			<td>Hausser Scientific</td>
			<td>3120</td>
			<td>Used to assess cell lysis</td>
		</tr>
		<tr>
			<td>JA 9.100 centrifuge rotor&#160;</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to harvest the yeast cells</td>
		</tr>
		<tr>
			<td>JA 20 fixed-angle centrifuge rotor</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to clear the cell extract of non-soluble cellular material</td>
		</tr>
		<tr>
			<td>Ti 60 fixed-angle centrifuge rotor</td>
			<td>Beckman Coulter, Inc.</td>
			<td></td>
			<td>Used to further clear the soluble cell extract</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>R5355</td>
			<td>Temperature controlled shaker</td>
		</tr>
		<tr>
			<td>Novex gel system</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IgG resin</td>
			<td>GE Healthcare</td>
			<td>17-0969-01</td>
			<td>Sepharose 6 fast flow</td>
		</tr>
		<tr>
			<td>Calmodulin resin</td>
			<td>Agilent Technologies, Inc.</td>
			<td>214303</td>
			<td>Affinity resin</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail, mini tablets</td>
			<td>Sigma Aldrich</td>
			<td>589297</td>
			<td>Mini cOmplete ultra EDTA-free tablets</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail, large tablets</td>
			<td>Sigma Aldrich</td>
			<td>5892953</td>
			<td>cOmplete ultra EDTA-free tablets</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td></td>
			<td></td>
			<td>Dissolved in isopropanol</td>
		</tr>
		<tr>
			<td>2 mL Bio-spin column</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>7326008</td>
			<td>Used to pack and wash the Calmodulin resin</td>
		</tr>
		<tr>
			<td>10 mL poly-prep column</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>7311550</td>
			<td>Used to pack and wash the IgG resin</td>
		</tr>
		<tr>
			<td>Precast native PAGE Bis-Tris gels</td>
			<td>Life Technologies</td>
			<td>BN1002</td>
			<td>Novex NativePAGE Bis-Tris 4-16% precast polyacrylamide gels</td>
		</tr>
		<tr>
			<td>NativeMark protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>LC0725</td>
			<td>Unstained protein standard used for native PAGE. Load 7.5 &#956;L for a silver stained gel and 5 &#956;L for a SYPRO Ruby stained gel</td>
		</tr>
		<tr>
			<td>Precast SDS PAGE Bis-Tris gels</td>
			<td>Life Technologies</td>
			<td>NP0321</td>
			<td>Novex Nu-PAGE Bis-Tris 4-12% precast polyacrylamide gels&#160;</td>
		</tr>
		<tr>
			<td>PageRuler protein standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>26614</td>
			<td>Unstained protein standard used for Western blotting</td>
		</tr>
		<tr>
			<td>SDS running buffer</td>
			<td>Life Technologies</td>
			<td>NP0001</td>
			<td>1x NuPAGE MOPS SDS Buffer</td>
		</tr>
		<tr>
			<td>TAP antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>CAB1001</td>
			<td>Primary antibody against CBP tag</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>31341</td>
			<td>Goat anti-rabbit alkaline phosphatase conjugated</td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>Thermo Fisher Scientific</td>
			<td>34042</td>
			<td>5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium&#160;</td>
		</tr>
		<tr>
			<td>Dialaysis units</td>
			<td>Thermo Fisher Scientific</td>
			<td>88401</td>
			<td>Slide-A-Lyzer mini dialysis units</td>
		</tr>
		<tr>
			<td>Centrifugal filter units, 100kDa MWCO</td>
			<td>EMD Millipore</td>
			<td>UFC5100008</td>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td>
		</tr>
		<tr>
			<td>Detergent absorbing beads</td>
			<td>Bio-Rad Laboratories, Inc.</td>
			<td>1523920</td>
			<td>Bio-bead SM-2 absorbants</td>
		</tr>
		<tr>
			<td>SYBR Green II</td>
			<td>Thermo Fisher Scientific</td>
			<td>S-7564</td>
			<td>Flourescent dye for nucleic acid staining, when detecting with SYPRO Ruby present, use excitation wavelength of 488 nm and emission wavelength of 532 nm</td>
		</tr>
		<tr>
			<td>SYPRO Ruby</td>
			<td>Molecular Probes</td>
			<td>S-12000</td>
			<td>Flourescent dye for protein staining, when detecting with SYBR Green II present, use excitation wavelength of 457 nm and emission wavelength of 670 nm</td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>G400-CP</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification of native complexes for structural study using a tandem affinity tag method

亲和纯化的方法已经成功地分离出原生复合蛋白质组学表征。结构的异质性和一定程度的复杂的成分异质性通常不妨碍进行这种研究的进展。与此相反，用于结构表征的复合应是既在组成和结构上均匀的，以及在比所需的蛋白质组学更高浓度的状态下进行纯化。最近，一直在电子显微镜的应用的大型大分子复合物结构的决心显著的进步。这加剧了利益的方法通过电子显微镜净化足够数量和质量结构测定天然复合物。串联亲和纯化（TAP）的方法进行了优化，提取和纯化的18亚基，从〜芽殖酵母0.8丙二醛核蛋白组件（ 酿酒酵母） </EM&gt;适用于负染色和电子显微镜低温。这里有详细的TAP方法，做出这些改变的理由所作的修改，以及采取的方法测定的在组成和结构均匀复杂。

使用了改良的方法TAP从S.净化酵母 U1的核蛋白，18亚基核糖核蛋白复合体。公布的协议2,3以下复杂的初始的TAP纯化，得到一个复合物，出现异类，迁移作为银三个频带染色天然聚丙烯酰胺凝胶（ 图2A）。在TAP方法的优化的多轮，产生了迁移，主要是一个单波段上指示更均匀组件（ 图2A）的天然凝胶的配合物。用荧光染料染色为...

Watch this Scientific Journal Video about Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method at JoVE.com

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

The overall goal of this protocol is to purify a native macromolecular complex of suitable quality for biophysical studies. This protocol serves not only as a way to purify a complex, but as a detailed guide to assessing the structural quality of the sample during purification. The main advantage of this protocol is that it allows one to purify a native assembly under near physiological buffer conditions simply and rapidly, so as to ensure compositional homogeneity.After centrifugation of S.cerevisiae cells, and decanting the supernatant according to the text protocol, add two milliliters of chilled lysis buffer, and swirl and/or use a 10 milliliter serelogical pipette to suspend the pellet. Transfer the cell suspension into an empty 50 milliliter conical polypropylene centrifuge tube kept on ice. Then use an additional two milliliters of chilled lysis buffer to wash each centrifuge bottle and add the solution to the 50 milliliter tube.After determining the wet weight of the cell pellet, create a liquid nitrogen bath in and around a 50 milliliter conical polypropylene centrifuge tube. Next, draw suspended cells into a five milliliter syringe and connect a 16 gauge needle to the syringe. Then, freeze the cell suspension by passing it through the syringe and needle into the bath at a rate of approximately one milliliter per minute to generate droplets.To lyse the yeast cells, begin by using liquid nitrogen to pre-chill a coffee grinder. Then add up to 40 grams of yeast cells and grind for 25 seconds, repeating the grinding eight or nine times. It is recommended not to purify for more than 10 liters of cell culture at one time.This minimizes purification time and ensures that structural integrity of the complex is maintained. After every two rounds of grinding, add a shallow layer of liquid nitrogen to the grinder and allow it to evaporate. To keep the cells from clumping, prevent the cells from thawing by using a liquid nitrogen chilled spatula to stir the cells.Following lysis, the cells should appear as a fine white powder. After checking cell lysis and preparing lysis buffer with PMSF, DTT and protease inhibitors according to the text protocol, use a liquid nitrogen chilled spatula to incrementally scoop the frozen lysed cell powder into the prepared 50 milliliter tubes of the buffer. With each added increment, gently rotate the 50 milliliter tubes at room temperature so as to thaw and dissolve the cells and to prevent bubbles from forming.As the cells dissolve, add more cell powder. After all the cells have been added, continue thawing and dissolving the cells until there are no frozen cell clumps observed in the tubes. This should take approximately 50 minutes.Next, centrifuge the cell lysate at 25, 000 times g and four degrees Celsius for 20 minutes. Once cells are lysed and following centrifugation, you may observe a small dark layer in the insoluble pellet. Transfer the supernatant to polycarbonate ultracentrifuge tubes and add 10 microliters of 200 millimolar PMSF to each full tube.Centrifuge the samples at 100, 000 times g and four degrees Celsius for one hour. At the end of the spin, four layers should be visible. A hard clear pellet containing mostly ribosomal complexes;a soft lipid rich pellet;a large, clear, yellow-tinged layer containing most of the cell's soluble proteins and complexes;and a scaly top layer consisting of lipids.In a cold room at four degrees Celsius, use a one milliliter pipette to remove as much of the top scaly lipid layer as possible and discard. Use a 10 milliliter serological pipette to recover most of the clear yellow layer. Then, using a one milliliter pipette to avoid disturbing the bottom two layers, recover the last few milliliters of this layer.From 40 grams of wet cell pellet, approximately 48 milliliters of the middle layer is typically recovered. After preparing IgG sepharose according to the text protocol, combine the IGG resin with the middle phase supernatant and two mini protease inhibitor tablets per 40 grams of cells. Then place on a rotator at four degrees Celsius for two hours.This is the IgG batch solution. Prepare two 10 milliliter polyprep columns by cutting the ends of the columns to produce a flat opening. Pour the 24 milliliters of suspend IgG resin slurry or batch solution into each column, and allow to sediment by gravity.This corresponds to 200 microliters of packed IgG resin. If sedimentation takes longer than 30 minutes, the middle phase may have been contaminated by lipids. When sedimentation is complete, use four successive 10 milliliter volumes of IgG D150 buffer to wash the packed column.To carry out tobacco etch virus or TEV protease cleavage on the column, seal the bottom of the column and add one milliliter of IgG D150 buffer and 100 microliters of TEV protease. Seal the top of the column and using a thermal mixer at 750 RPM and 18 degrees Celsius, mix for 20 minutes. Re-suspend the resin and mix again for 20 minutes.Then re-suspend and repeat the 20 minute mixing a third time. Return the column to four degrees Celsius and use gravity to elute the protein complex. Then, add 200 microliters of IgG D150 to elute the dead volume.After preparing calmodulin affinity resin according to the text protocol, add the calmodulin binding buffer and sample to the resin, and use a tube rotator to incubate at four degrees Celsius for one hour. Prepare a two milliliter polyprep column per 100 microliters of calmodulin slurry by cutting the end of the column to create a flat opening. The protocol details specific buffer and residence volumes chosen so as to keep the complex concentrated and to minimize its resin residence time.If cultural volume is changed, it is recommended to change the gravity columns'resin and buffer volumes to account for the change. Pack the column with no more than 100 microliters of calmodulin slurry and by gravity, use 5 milliliters of calmodulin binding buffer to wash the resin three times. Using 200 microliters of calmodulin elution buffer, elute the protein three times.Then, seal the bottom of the column and add 20 microliters of elution buffer and incubate for 2.5 minutes before releasing the seal and allowing the protein to elute by gravity. Seal the bottom of the column again and add 200 microliters of elution buffer before incubating for five minutes. Then, unseal the bottom of the column and elute the solution.For the sixth and final elution, seal the bottom of the column and incubate with elution buffer for 10 minutes. Then, unseal and elute. Carry out post-purification analysis and store samples according to the text protocol.As shown here, an initial TAP purification of the complex following a published protocol yielded a complex that migrated as three bands on a silver-stained gel. Multiple rounds of optimization of the TAP method gives us a complex that migrated as a single band on a native gel, indicative of a more homogeneous assembly. Consistent with the native gel result, western blotting using a TAP antibody against the complex, purified according to the published protocol, detected proteolysis of the TAP tagged protein, SNU-71.Modifications of the TAP method significantly reduced the amount of proteolysis, as nearly all of the 17 proteins in the U1 snRNP complex were resolved by SDS-PAGE and positively identified by multi mass spectrometry. Negative stain electron microscopy shows monodispersed particles from a successful purification after the first and second steps of TAP. In contrast, no particles of the correct size are observed when the purification is unsuccessful.Final complex quality was assessed on a lawn of purified particles, shown here, that appear monodispersed in the negative stain EM raw image. In addition, class averages of the particles reveal distinct features indicating that the sample is possibly suitable for structural study. Once mastered, this protocol can be completed within 10 to 12 hours of thawing the frozen lysed cell powder.While following this protocol, it's important to ensure that all solutions are free of proteases and nucleases, as well as to work rapidly to prevent aggregation or dissociation of the complex. After watching this video, you should understand how to purify a native complex of suitable quality for structural study, as well as how to assess that this has been achieved.