基于免疫沉淀的技术：使用阿加玫瑰珠纯化内源性蛋白质
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Overview
资料来源：苏珊娜·希斯勒1，托尼娅·韦伯1
1马里兰大学巴尔的摩分校微生物学和免疫学系，MD 21201
免疫沉淀（IP，也称为”下拉”测定）是一种广泛使用的技术，在各种领域都有应用。它于1984年首次构思，1988年经过提炼（1，2）。IP 的基本目标是使用针对该蛋白质的抗体对特定蛋白质进行纯化和分离。”免疫”一词是指使用抗体，而”沉淀”一词是指从溶液中拉出特定物质。目标蛋白可能是内源性或重组蛋白。大多数重组蛋白都有一个表位标记（即myc或标志）附着在它们上，以简化随后的纯化。通常，优化重组蛋白 IP 更容易，因为针对重组表位标记的抗体非常强和有效。针对内源性蛋白质的抗体具有极其可变的疗效 – 使得优化这些 IP 更加困难。免疫沉淀后的一个必要步骤是纯化验证。分离的蛋白质使用SDS-PAGE解决，随后由西方血带探查纯度（图1）。一个重要的控制是在西方印块期间使用不同的抗体来验证正确蛋白质的提取。IP 与后续技术的结合是一种强大的分析工具。纯化后的目标可能是通过NMR、质谱和体外测定对蛋白质本身进行表征，或分析蛋白质的相互作用伙伴（即蛋白质、DNA、RNA）（3、4、5）。
图1：免疫沉淀程序概述。免疫沉淀是使用抗体分离特定蛋白质。从细胞中生产分解后，有两个主要步骤-预清除和拉下。在预清除步骤中，细胞解物预先清除使用等型对照抗体与非特定抗体结合的蛋白质。在下拉步骤中，使用蛋白质特异性抗体将目标蛋白拉下。分离的蛋白质然后由西方斑点进行分析。等型抗体和蛋白质特异性抗体具有相同的恒定域，但抗原结合域不同。该协议的一个关键成分是蛋白质A/G腺珠，它结合抗体的恒定域-允许目标蛋白的免疫沉淀。请点击此处查看此图的较大版本。
抗体是免疫沉淀的关键成分，它区别于其他形式的蛋白质纯化（即镍亲基柱纯化）。抗体是由B细胞制成的分子，可以识别特定的蛋白质表皮。抗体有两个域：常数（Fc）和抗原结合（Fab）（图1）。常量域识别抗体的类型，并指示体内的功能。通常，用于IP的抗体的恒定域是小鼠、大鼠或兔子IgG。抗体的抗原结合部分识别特定蛋白质的特定表位。抗体可以识别折叠蛋白上的缩影，当蛋白质变性时，这些蛋白质可能不存在，反之亦然。因此，表位的可用性取决于蛋白质折叠 – 识别在选择抗体和IP条件时要考虑的重要因素。
原核素和真核系统都有抗体结合蛋白。在真核系统中，目的是免疫保护细菌，而在原核系统，目的是保护免疫系统。抗体结合蛋白以两种方式影响 IP 方法。首先，有必要的预清除步骤（图1）去除结合抗体的蛋白质的解结-从而减少最终产品中的非特异性结合。此步骤使用同型抗体，其恒定域与蛋白质特异性抗体不同，但抗体结合域不同。细菌抗体结合蛋白是该方法的第二个关键成分。在蛋白质特异性抗体结合靶蛋白后，抗体：蛋白质复合物必须被拉下（图1）。蛋白质A、G和L是结合抗体恒定领域的细菌蛋白。虽然细菌用它来破坏免疫系统，研究人员已经加入这个系统，以方便抗体纯化，它用于预清除和下拉步骤。这些蛋白质对于不同的物种和不同的恒定域亚型有不同的结合亲和力-在选择知识产权条件时需要考虑的另一个因素。许多公司销售标有A/G标记的甘蔗珠（图1）、预制旋转柱或树脂来制造柱。通常，珠子和旋转柱用于较小的样品尺寸，而树脂用于散装纯化。
在本实验练习中，我们演示如何使用基于蛋白质 A/G Plus 的腺苷酸腺苷酸细胞从原生鼠胸腺细胞中纯化内源性蛋白 c-myc。该协议从细胞分解液制备开始，最后通过西方斑点分析验证成功的蛋白质提取。
Procedure
Results
The results of the procedure detailed above are shown in Figure 2. From left to right, the lanes contain the control group (isotype), the test group (c-myc), the pre-cleared lysate (lysate), and the molecular weight ladder (ladder). The 25 and 75 kDa ladder bands are marked. The two prominent bands at ~25 kDa and 50 kDa are the light and heavy chain of the binding antibody, respectively and are non-specific to the IP or the samples. c-myc protein which runs around 67kDa on Western blots and is usually visible just below the 75 kDa ladder band. In this blot, the c-myc band is visible in the second lane, but absent in the first lane, indicating that the IP antibody successfully pulled down c-myc. There is no visible band in the pre-cleared lysate lane, suggesting that this protein has low endogenous expression levels.
Figure 2: Results of a Western Blot Analysis, used to assess the purification of c-myc by immunoprecipitation. A band at 67 kDa, corresponding to c-myc, is visible in the anti-c-myc lane, but not the isotype control lane. Note that c-myc levels were not high enough to be visualized in the lysate lane. Please click here to view a larger version of this figure.
Applications and Summary
In short, immunoprecipitation is the isolation of a specific protein using an antibody. In this example, the results of the immunoprecipitation were analyzed by Western blot to assess the purity. The isolated protein could be used in a number of applications afterwards including: NMR for protein structure, Mass Spectrometry for amino acid sequence, or in vitro assays for enzymatic characterization. IPs can also characterize the interacting partners of proteins. For instance, following isolation, DNA or RNA could be isolated for sequencing. Co-immunoprecipitations assess protein-protein interactions. When the target protein is pulled down during an IP, interacting proteins can also be pulled down. These interacting partners can be assessed by mass spectrometry and Western blot. Immunoprecipitation is a powerful technique for studying protein biology.
References
- Olliver, C. L. and Boyd, C. D. (1984). Immunoprecipitation of In Vitro Translation Products with Protein A Bound to Sepharose. In J. M. Walker (eds), Nucleic Acids. Methods in Molecular Biology (pp. 157-160). New Jersey: Humana Press.
- Thurston, C. F. and Henley, L. F. (1988). Direct Immunoprecipitation of Protein. In J. M. Walker (eds), New Protein Techniques. Methods in Molecular Biology (pp. 149-158). New Jersey: Humana Press.
- Anderson, N. G. (1998). Co-immunoprecipitation: Identification of Interacting Proteins. In R. A. Clegg (eds), Protein Targeting Protocols.Methods in Molecular Biology (pp. 35-45). New Jersey: Humana Press.
- Jackson, D. I. and Dickson, C. (1999). Protein Techniques: Immunoprecipitation, In Vitro Kinase Assays, and Western Blotting. In P.T. Sharpe and I. Mason (eds), Molecular Embryology. Methods in Molecular Biology (pp. 699-708). New Jersey: Humana Press.
- Trieu, E. P. and Targoff, I. N. (2015). Immunoprecipitation: Western Blot for Proteins of Low Abundance. In B. Kurien and R. Scofield (eds), Western Blotting. Methods in Molecular Biology (pp. 327-342). New York, NY: Humana Press.
Transcript
Immunoprecipitation, or IP, is a widely used technique to isolate a protein of interest from a cell or tissue lysate or a body fluid for protein characterization or to investigate protein-protein interactions.
The process begins with an antibody, which has a high affinity and specificity for the target protein. This antibody is mixed with the sample, allowing antibody-target complexes to form. Any protein bound to the target protein also gets indirectly attached to the antibody in the process. Next, the solution is incubated with agarose beads, conjugated to a bacterial protein, which has a strong affinity for the constant region of antibodies. The bacterial protein binds to the antibody and connects the antibody- target complexes to the beads. Then, the solution is centrifuged to precipitate the beads, thereby extracting the entire complex containing the binding antibody, the target protein, and any interacting proteins. Finally, the bound proteins are extracted from the beads and released from each other and are used for further analysis by techniques such as Western blotting.
Several variations of different parts of this technique are commonly used, like pre-clearing, using peptide tags or magnetic beads, or analyzing other non-protein binding partners. IP can be preceeded by a pre-clearing step, to remove non-specific antibody-binding proteins in the sample and minimize background. This involves first incubating the sample with isotype control antibodies, allowing them to bind to these proteins, and then using agarose beads to precipitate the complexes. The sample is then ready to proceed to the actual IP.
Peptide tags are useful if a specific antibody is not available for IP. Here, the target protein can be genetically modified to contain a peptide epitope tag and an antibody against the tag is able to pull out the protein of interest. Magnetic beads are often used instead of agarose to precipitate the target. After binding to the antibody-target complex, the sample tube is placed in a strong magnetic field, which extracts the beads from the solution. This eliminates the need for centrifugation and improves speed and convenience.
Immunoprecipitation is also used for studying DNA or RNA binding proteins and are known as chromatin immunoprecipitation and RNA immunoprecipitation, respectively. These variations are useful for troubleshooting and adapting the method for different experimental applications. In this video, you will observe how to pre-clear a cell lysate and perform immunoprecipitation to extract a protein of interest, followed by Western blot analysis to validate the experiment.
To begin, place the pre-collected cells in a microcentrifuge and spin at 13 thousand rpm for three minutes. Following the spin, remove the supernatant and then resuspend the cells in 500 microliters of lysis buffer RIPA with PMSF. Now, disrupt the cells using a few quick pulses with a vortex and then aspirate the lysate a few times with a 25 gauge needle attached to a syringe, taking care to avoid creating bubbles. Place the cells on ice for 15 minutes. After incubating the samples on ice, centrifuge the lysate for 15 minutes at four degrees celsius.
Label a new 1.5 milliliter microcentrifuge tube. Following the spin, transfer the supernatant to the freshly labeled tube and discard the pellet. Next, pre-clear the lysate of contaminants that bind non-specifically to either the agarose beads or the primary antibody by adding 20 microliters of the Protein A/G PLUS-agarose beads and one microgram of an isotype control antibody to the lysate, which in this example is a mouse IgG1 isotype control. Incubate the tube on a rotator in a cold room for 30 minutes. After rotating the lysate in the cold room for 30 minutes, centrifuge the sample at 3200 rpm for 30 seconds at four degrees celsius. Remove the tube from the centrifuge and transfer the pre-cleared supernatant to a fresh labeled 1.5 milliliter microcentrifuge tube. Discard the pellet.
Now, determine the protein concentration of the cell lysate by performing a Bradford assay. Label seven 1. 5-milliliter microcentrifuge tubes one through six and sample and aliquot 1000 microliters of the Bradford reagent into each tube. Six of the tubes will be used to make a standard curve by adding various amounts of known quantities of BSA to each tube. The amounts to add are listed in this table. In the seventh sample tube, add one microliter of the pre-cleared lysate. Place 200 microliters from each of the seven tubes into individual wells of a flat-bottom 96-well plate, repeating each sample in triplicate so that there are three columns of seven samples. Read the plate on a plate reader, using a wavelength of 595 nanometers. After creating a standard curve in Excel, calculate the protein concentration of the pre-cleared lysate.
Next, label two 1.5-milliliter microcentrifuge tubes- one as control and the other as test, which in this example, will be the c-myc antibody. Place 500 micrograms of the pre-cleared lysate into each of these tubes and then bring the total volume for each tube up to 500 microliters using lysis buffer. Next, add two micrograms of the anti-c-myc antibody to the test group tube. For the control, add two micrograms of the mouse IgG1 isotype control antibody. Once the antibodies are added to the tubes, place the samples on a rotator in a cold room and incubate for two hours. Now, add the agarose beads. To do this, it is recommended to cut off the end of a pipette tip and then, using this modified tip, add 200 microliters of the Protein A/G PLUS-agarose beads to each tube. Incubate the tubes on a rotator in the cold room overnight.
Following the incubation, remove the tubes from the rotator and spin the lysates in the microcentrifuge to pull down the beads. After the spin is complete, remove the tubes from the centrifuge and aspirate the supernatant from each tube. Next, wash the beads using 500 microliters of 1X Dulbecco’s PBS. Place the tubes in a microcentrifuge and spin down for 30 seconds at four degrees celsius. Following this, remove the supernatant. Repeat the wash and centrifuge steps one more time for a total of two times. Remove the tubes from the microcentrifuge and aspirate the buffer from each tube. Using gel loading tips, remove any left over buffer from the beads, keeping the beads on ice to elute the bound protein.
In this example, the protein is eluted into SDS-PAGE running buffer by boiling for Western blot analysis. To do this, resuspend the beads in 20 microliters of SDS-PAGE loading dye containing beta-mercaptoethanol, or BME. Boil the samples at 95 degrees celsius for five minutes to dissociate the immunocomplexes from the beads. Then, centrifuge the beads at maximum speed for 10 seconds at room temperature. Remove the tubes from the microcentrifuge and hold them in a rack at room temperature. Using gel loading tips, carefully pipette the samples from the beads and load them into wells of a 4 to 15% gradient SDS-PAGE gel. In addition to the samples, load a lane with a protein ladder as well as a lane with the pre-cleared lysate to serve as a loading control. Once the gel is loaded, run the gel at 100 volts.
After the dye front has reached the bottom of the gel, which should take approximately one hour, stop the gel and make a Western blot sandwich, ensuring that the PVDF membrane is between the gel and the cathode. Place the Western blot sandwich in the transfer apparatus and transfer the proteins on the gel to the membrane for one hour at 100 volts. After the transfer is complete, place the membrane in five milliliters of block to prevent the antibodies from binding non-specifically to the membrane. Rock at a low setting for an hour at room temperature. When the timer sounds, remove the blocking buffer. Add five milliliters of the blocking buffer with the detection antibody to the membrane. Here, an anti-c-myc antibody, that is different than the one used for the pull down, is used.
Incubate the blot over night, at four degrees celsius on a rocker at a low setting. Following the incubation, remove the antibody and blocking buffer. Wash the blot, using five milliliters of TBST for five minutes at room temperature, on a rocker at a low setting. This wash step should be repeated two to five times for a total of three to six washes, using fresh TBST for each wash. Add five milliliters of one to 1000 secondary antibody and blocking buffer to the blot. In this case, the secondary antibody is HRP-tagged anti-rabbit light chain. Incubate the blot on a rocker at a low setting for one our at room temperature. Next, remove the buffer and wash the blot with five milliliters of TBST. Incubate this wash on a rocker at a low setting for five minutes at room temperature. Repeat this wash for a total of six to 12 washes, each with a fresh five milliliters of TBST. Remove the final wash by first pouring the liquid off of the blot. Then, using tweezers, dab the edge of the blot on a laboratory wipe to remove any excess liquid and then place the blot in a fresh container. Next, cover the blot with 1X Chemiluminescent Detection Reagent and incubate for one minute.
Working quickly, dab the edge of the blot on a laboratory wipe to remove any excess detection reagent and then place the blot on the imaging surface of the Imager tray. Image using the Chemiluminescent program to capture multiple time points from 10 to 30 seconds. After the blot is imaged, choose an image with optimal band visibility and then export that image. Prior to moving the blot, use the Imager to take a picture of the blot to capture the location of the ladder. Then, export that image also. Finally, using a slide preparation software, such as PowerPoint, align the bands and ladder images to form one image.
This image shows the Western blot result for immunoprecipitation of the protein c-myc from thymocyte cells. From left to right, the lanes represent the isotype control, the c-myc IP, and the pre-cleared lysate input. The lane on the extreme right is a merged image of the molecular weight ladder. The strong band, at around 25 kilodaltons is from the light chain and the one at 50 kilodaltons is from the heavy chain of the binding antibody and are non-specific to the IP or the samples. C-myc runs around 67 kilodaltons on Western blots and is usually visible just below the 75 kilodalton ladder band. In this blot, the c-myc band is visible in the second lane but absent in the first lane, indicating that the IP antibody successfully pulled down c-myc. There is no visible band in the pre-cleared lysate lane, suggesting that this protein has low endogenous expression levels.