486:
basic preclearing procedure is described below, wherein the lysate is incubated with beads alone, which are then removed and discarded prior to the immunoprecipitation. This approach, though, does not account for non-specific binding to the IP antibody, which can be considerable. Therefore, an alternative method of preclearing is to incubate the protein mixture with exactly the same components that will be used in the immunoprecipitation, except that a non-target, irrelevant antibody of the same antibody subclass as the IP antibody is used instead of the IP antibody itself. This approach attempts to use as close to the exact IP conditions and components as the actual immunoprecipitation to remove any non-specific cell constituent without capturing the target protein (unless, of course, the target protein non-specifically binds to some other IP component, which should be properly controlled for by analyzing the discarded beads used to preclear the lysate). The target protein can then be immunoprecipitated with the reduced risk of non-specific binding interfering with data interpretation.
472:/agarose beads is not completely saturated with antibodies. It often happens that the amount of antibody available to the researcher for their immunoprecipitation experiment is less than sufficient to saturate the agarose beads to be used in the immunoprecipitation. In these cases the researcher can end up with agarose particles that are only partially coated with antibodies, and the portion of the binding capacity of the agarose beads that is not coated with antibody is then free to bind anything that will stick, resulting in an elevated background signal due to non-specific binding of lysate components to the beads, which can make data interpretation difficult. While some may argue that for these reasons it is prudent to match the quantity of agarose (in terms of binding capacity) to the quantity of antibody that one wishes to be bound for the immunoprecipitation, a simple way to reduce the issue of non-specific binding to agarose beads and increase specificity is to preclear the lysate, which for any immunoprecipitation is highly recommended.
539:
immunoprecipitating extremely large protein complexes because of the complete lack of an upper size limit for such complexes, although there is no unbiased evidence stating this claim. The nature of magnetic bead technology does result in less sample handling due to the reduced physical stress on samples of magnetic separation versus repeated centrifugation when using agarose, which may contribute greatly to increasing the yield of labile (fragile) protein complexes. Additional factors, though, such as the binding capacity, cost of the reagent, the requirement of extra equipment and the capability to automate IP processes should be considered in the selection of an immunoprecipitation support.
518:, exhibit exact uniformity, and therefore all beads exhibit identical physical characteristics, including the binding capacity and the level of attraction to magnets. Polydisperse beads, while similar in size to monodisperse beads, show a wide range in size variability (1 to 4 ÎĽm) that can influence their binding capacity and magnetic capture. Although both types of beads are commercially available for immunoprecipitation applications, the higher quality monodisperse superparamagnetic beads are more ideal for automatic protocols because of their consistent size, shape and performance. Monodisperse and polydisperse superparamagnetic beads are offered by many companies, including
447:(also known as agarose resins or slurries). The advantage of this technology is a very high potential binding capacity, as virtually the entire sponge-like structure of the agarose particle (50 to 150 ÎĽm in size) is available for binding antibodies (which will in turn bind the target proteins) and the use of standard laboratory equipment for all aspects of the IP protocol without the need for any specialized equipment. The advantage of an extremely high binding capacity must be carefully balanced with the quantity of antibody that the researcher is prepared to use to coat the agarose beads. Because antibodies can be a cost-limiting factor, it is best to calculate backward
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221:
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beads per IP). This is because sepharose beads must be concentrated at the bottom of the tube by centrifugation and the supernatant removed after each incubation, wash, etc. This imposes absolute physical limitations on the process, as pellets of agarose beads less than 25 to 50 ÎĽl are difficult if not impossible to visually identify at the bottom of the tube. With magnetic beads, there is no minimum quantity of beads required due to magnetic handling, and therefore, depending on the target antigen and IP antibody, it is possible to use considerably less magnetic beads.
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specifically recognize. Once this has occurred the immunoprecipitation portion of the protocol is actually complete, as the specific proteins of interest are bound to the antibodies that are themselves immobilized to the beads. Separation of the immunocomplexes from the lysate is an extremely important series of steps, because the protein(s) must remain bound to each other (in the case of co-IP) and bound to the antibody during the wash steps to remove non-bound proteins and reduce background.
635:
and higher recoveries, the process is often performed in small spin columns with a pore size that allows liquid, but not agarose beads, to pass through. After centrifugation, the agarose beads will form a very loose fluffy pellet at the bottom of the tube. The supernatant containing contaminants can be carefully removed so as not to disturb the beads. The wash buffer can then be added to the beads and after mixing, the beads are again separated by centrifugation.
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beads due to the large bead size and sponge-like structure. But the variable pore size of the agarose causes a potential upper size limit that may affect the binding of extremely large proteins or protein complexes to internal binding sites, and therefore magnetic beads may be better suited for immunoprecipitating large proteins or protein complexes than agarose beads, although there is a lack of independent comparative evidence that proves either case.
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the magnet) with the washing solution and then concentrating the beads back on the tube wall (by placing the tube back on the magnet). The washing is generally repeated several times to ensure adequate removal of contaminants. If the superparamagnetic beads are homogeneous in size and the magnet has been designed properly, the beads will concentrate uniformly on the side of the tube and the washing solution can be easily and completely removed.
25:
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additional members). By repeating the immunoprecipitation in this way, the researcher verifies that each identified member of the protein complex was a valid identification. If a particular protein can only be recovered by targeting one of the known members but not by targeting other of the known members then that protein's status as a member of the complex may be subject to question.
285:. At this point the immunoprecipitation is performed resulting in the purification of protein–DNA complexes. The purified protein–DNA complexes are then heated to reverse the formaldehyde cross-linking of the protein and DNA complexes, allowing the DNA to be separated from the proteins. The identity and quantity of the DNA fragments isolated can then be determined by
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agarose beads, which would obviously be an economical disadvantage of using agarose. While these arguments are correct outside the context of their practical use, these lines of reasoning ignore two key aspects of the principle of immunoprecipitation that demonstrates that the decision to use agarose or magnetic beads is not simply determined by binding capacity.
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Using the traditional batch method of immunoprecipitation as listed below, where all components are added to a tube during the IP reaction, the physical handling characteristics of agarose beads necessitate a minimum quantity of beads for each IP experiment (typically in the range of 25 to 50 ÎĽl
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Proponents of both agarose and magnetic beads can argue whether the vast difference in the binding capacities of the two beads favors one particular type of bead. In a bead-to-bead comparison, agarose beads have significantly greater surface area and therefore a greater binding capacity than magnetic
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the lysate at the start of each immunoprecipitation experiment (see step 2 in the "protocol" section below) is a way to remove potentially reactive components from the cell lysate prior to the immunoprecipitation to prevent the non-specific binding of these components to the IP beads or antibody. The
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onto either the C- or N- terminal end of the protein of interest. The advantage here is that the same tag can be used time and again on many different proteins and the researcher can use the same antibody each time. The advantages with using tagged proteins are so great that this technique has become
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With superparamagnetic beads, the sample is placed in a magnetic field so that the beads can collect on the side of the tube. This procedure is generally complete in approximately 30 seconds, and the remaining (unwanted) liquid is pipetted away. Washes are accomplished by resuspending the beads (off
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When working with agarose beads, the beads must be pelleted out of the sample by briefly spinning in a centrifuge with forces between 600–3,000 x g (times the standard gravitational force). This step may be performed in a standard microcentrifuge tube, but for faster separation, greater consistency
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While clear benefits of using magnetic beads include the increased reaction speed, more gentle sample handling and the potential for automation, the choice of using agarose or magnetic beads based on the binding capacity of the support medium and the cost of the product may depend on the protein of
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An indirect approach is sometimes preferred when the concentration of the protein target is low or when the specific affinity of the antibody for the protein is weak. The indirect method is also used when the binding kinetics of the antibody to the protein is slow for a variety of reasons. In most
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Antibodies that are specific for a particular protein, or a group of proteins, are added directly to the mixture of protein. The antibodies have not been attached to a solid-phase support yet. The antibodies are free to float around the protein mixture and bind their targets. As time passes, beads
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Proponents of magnetic beads claim that the beads exhibit a faster rate of protein binding over agarose beads for immunoprecipitation applications, although standard agarose bead-based immunoprecipitations have been performed in 1 hour. Claims have also been made that magnetic beads are better for
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Second, the ability to capture the target protein is directly dependent upon the amount of immobilized antibody used, and therefore, in a side-by-side comparison of agarose and magnetic bead immunoprecipitation, the most protein that either support can capture is limited by the amount of antibody
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As successive rounds of targeting and immunoprecipitations take place, the number of identified proteins may continue to grow. The identified proteins may not ever exist in a single complex at a given time, but may instead represent a network of proteins interacting with one another at different
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beads for immunoprecipitation is a newer approach that is gaining in popularity as an alternative to agarose beads for IP applications. Unlike agarose, magnetic beads are solid and can be spherical, depending on the type of bead, and antibody binding is limited to the surface of each bead. While
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Lysates are complex mixtures of proteins, lipids, carbohydrates and nucleic acids, and one must assume that some amount of non-specific binding to the IP antibody, Protein A/G or the beaded support will occur and negatively affect the detection of the immunoprecipitated target(s). In most cases,
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Once the solid substrate bead technology has been chosen, antibodies are coupled to the beads and the antibody-coated-beads can be added to the heterogeneous protein sample (e.g. homogenized tissue). At this point, antibodies that are immobilized to the beads will bind to the proteins that they
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Conversely, spin columns may be employed instead of normal microfuge tubes to significantly reduce the amount of agarose beads required per reaction. Spin columns contain a filter that allows all IP components except the beads to flow through using a brief centrifugation and therefore provide a
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Some argue that the significantly greater binding capacity of agarose beads may be a disadvantage because of the larger capacity of non-specific binding. Others may argue for the use of magnetic beads because of the greater quantity of antibody required to saturate the total binding capacity of
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As mentioned above, only standard laboratory equipment is required for the use of agarose beads in immunoprecipitation applications, while high-power magnets are required for magnetic bead-based IP reactions. While the magnetic capture equipment may be cost-prohibitive, the rapid completion of
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First, non-specific binding is not limited to the antibody-binding sites on the immobilized support; any surface of the antibody or component of the immunoprecipitation reaction can bind to nonspecific lysate constituents, and therefore nonspecific binding will still occur even when completely
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Repeating the experiment by targeting different members of the protein complex allows the researcher to double-check the result. Each round of pull-downs should result in the recovery of both the original known protein as well as other previously identified members of the complex (and even new
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This works when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple members of the complex out of the solution by latching onto one member with an antibody. This concept of pulling protein complexes out of solution is sometimes referred to as a
654:, or any number of other methods for identifying constituents in the complex. Protocol times for immunoprecipitation vary greatly due to a variety of factors, with protocol times increasing with the number of washes necessary or with the slower reaction kinetics of porous agarose beads.
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these beads do not have the advantage of a porous center to increase the binding capacity, magnetic beads are significantly smaller than agarose beads (1 to 4 ÎĽm), and the greater number of magnetic beads per volume than agarose beads collectively gives magnetic beads an effective
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immunoprecipitations using magnetic beads may be a financially beneficial approach when grants are due, because a 30-minute protocol with magnetic beads compared to overnight incubation at 4 °C with agarose beads may result in more data generated in a shorter length of time.
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the quantity of agarose that is needed to bind that particular quantity of antibody. In cases where antibody saturation is not required, this technology is unmatched in its ability to capture extremely large quantities of captured target proteins. The caveat here is that the
289:(PCR). The limitation of performing PCR on the isolated fragments is that one must have an idea which genomic region is being targeted in order to generate the correct PCR primers. Sometimes this limitation is circumvented simply by cloning the isolated genomic DNA into a
333:, cells are UV crosslinked prior to lysis, followed by additional purification steps beyond standard immunoprecipitation, including partial RNA fragmentation, high-salt washing, SDS-PAGE separation and membrane transfer, and identification of direct RNA binding sites by
178:(i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this
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From this point on, the direct and indirect protocols converge because the samples now have the same ingredients. Both methods give the same end-result with the protein or protein complexes bound to the antibodies which themselves are immobilized onto the beads.
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that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. Immunoprecipitation requires that the antibody be coupled to a solid
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exposed, thus failing to identify any proteins in complexes that hide the epitope. This can be seen in that it is rarely possible to precipitate even half of a given protein from a sample with a single antibody, even when a large excess of antibody is
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beads may make the sepharose beads appear less expensive. But magnetic beads may be competitively priced compared to agarose for analytical-scale immunoprecipitations depending on the IP method used and the volume of beads required per IP reaction.
401:(non-magnetic) beads. The beads with bound antibodies are then added to the protein mixture, and the proteins that are targeted by the antibodies are captured onto the beads via the antibodies; in other words, they become immunoprecipitated.
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Incubate solution with antibody against the protein of interest. Antibody can be attached to solid support before this step (direct method) or after this step (indirect method). Continue the incubation to allow antibody-antigen complexes to
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Wash precipitated complex several times. Spin each time between washes when using agarose beads or place tube on magnet when using superparamagnetic beads and then remove the supernatant. After the final wash, remove as much supernatant as
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An added benefit of using magnetic beads is that automated immunoprecipitation devices are becoming more readily available. These devices not only reduce the amount of work and time to perform an IP, but they can also be used for
740:
Rashid, K. A., Hevi, S., Chen, Y., Le Cahérec, F., & Chuck, S. L. (2002). A Proteomic
Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B. The Journal of Biological Chemistry, 277(24), 22010–22017.
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tag. While the use of a tag to enable pull-downs is convenient, it raises some concerns regarding biological relevance because the tag itself may either obscure native interactions or introduce new and unnatural interactions.
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The price of using either type of support is a key determining factor in using agarose or magnetic beads for immunoprecipitation applications. A typical first-glance calculation on the cost of magnetic beads compared to
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One of the major technical hurdles with immunoprecipitation is the great difficulty in generating an antibody that specifically targets a single known protein. To get around this obstacle, many groups will engineer
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to the cells (or tissue), although it is sometimes advantageous to use a more defined and consistent crosslinker such as dimethyl 3,3′-dithiobispropionimidate-2 HCl (DTBP). Following crosslinking, the cells are
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is used and has recently emerged as a standard technology that can localize protein binding sites in a high-throughput, cost-effective fashion, allowing also for the characterization of the
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to the DNA that they are binding. By using an antibody that is specific to a putative DNA binding protein, one can immunoprecipitate the protein–DNA complex out of cellular lysates. The
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the amount of antibody that is required to bind that quantity of protein (with a small excess added in order to account for inefficiencies of the system), and back still further
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Pre-clear the sample by passing the sample over beads alone or bound to an irrelevant antibody to soak up any proteins that non-specifically bind to the IP components.
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and then using primers that are specific to the cloning region of that vector. Alternatively, when one wants to find where the protein binds on a genome-wide scale,
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interest and the IP method used. As with all assays, empirical testing is required to determine which method is optimal for a given application.
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are added to the mixture of antibody and protein. At this point, the antibodies, which are now bound to their targets, will stick to the beads.
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756:"RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts"
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to isolate that particular protein out of a solution containing many different proteins. These solutions will often be in the form of a
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commonplace for all types of immunoprecipitation including all of the types of IP detailed above. Examples of tags in use are the
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1029:
Alber F, Dokudovskaya S, Veenhoff LM, et al. (November 2007). "Determining the architectures of macromolecular assemblies".
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Antibodies that are specific for a particular protein (or group of proteins) are immobilized on a solid-phase substrate such as
61:
1759:
1131:"The nuclear pore complex–associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly"
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SDS-PAGE followed by: gel staining, cutting out individual stained protein bands, and sequencing the proteins in the bands by
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Alber F, Dokudovskaya S, Veenhoff LM, et al. (November 2007). "The molecular architecture of the nuclear pore complex".
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325:, the co-purified RNAs are extracted and their enrichment is compared to control, which was originally done by microarray or
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added. So the decision to saturate any type of support depends on the amount of protein required, as described above in the
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saturated beads are used. This is why it is important to preclear the sample before the immunoprecipitation is performed.
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Historically the solid-phase support for immunoprecipitation used by the majority of scientists has been highly-porous
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member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify
917:"C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI"
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of interest. This technique gives a picture of the protein–DNA interactions that occur inside the nucleus of living
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The two general methods for immunoprecipitation are the direct capture method and the indirect capture method.
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nature of this method is in contrast to other approaches traditionally employed to answer the same questions.
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of a plant or animal tissue. Other sample types could be body fluids or other samples of biological origin.
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using another antibody for proteins that were interacting with the antigen, followed by detection using a
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the amount of protein that needs to be captured (depending upon the analysis to be performed downstream),
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RIP and CLIP both purify a specific RNA-binding protein in order to identify bound RNAs, thereby studying
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Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaz; Darnell, Robert B. (2003-11-14).
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190:"pull-down". Co-IP is a powerful technique that is used regularly by molecular biologists to analyze
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While the vast majority of immunoprecipitations are performed with agarose beads, the use of
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A particular antibody often selects for a subpopulation of its target protein that has the
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Bonifacino, J. S., Dell'Angelica, E. C. and
Springer, T. A. 2001. Immunoprecipitation.
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Commercially available magnetic beads can be separated based by size uniformity into
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Analyze complexes or antigens of interest. This can be done in a variety of ways:
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Elute proteins from the solid support using low-pH or SDS sample loading buffer.
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The principle underpinning this assay is that DNA-binding proteins (including
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Niepel M, Strambio-de-Castillia C, Fasolo J, Chait BT, Rout MP (July 2005).
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situations, the direct method is the default, and the preferred, choice.
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After washing, the precipitated protein(s) are eluted and analyzed by
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Crowell RE, Du Clos TW, Montoya G, Heaphy E, Mold C (November 1991).
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Precipitate the complex of interest, removing it from bulk solution.
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method to use significantly less agarose beads with minimal loss.
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that is manifested when the enormous binding capacity of the
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Cristea IM, Williams R, Chait BT, Rout MP (December 2005).
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806:"CLIP identifies Nova-regulated RNA networks in the brain"
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and the DNA is broken into pieces 0.2–1.0 kb in length by
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Involves using an antibody that is specific for a known
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Protein analysis and purification: benchtop techniques
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Lyse cells and prepare sample for immunoprecipitation.
231:(ChIP) is a method used to determine the location of
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49:. Unsourced material may be challenged and removed.
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754:Keene JD, Komisarow JM, Friedersdorf MB (2006).
1236:Introduction to Immunoprecipitation Methodology
1210:Analysis of Proteins Using Immunoprecipitation
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701:matrix-assisted laser desorption/ionization
170:Protein complex immunoprecipitation (Co-IP)
154:Individual protein immunoprecipitation (IP)
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1087:"Fluorescent proteins as proteomic probes"
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1228:at the U.S. National Library of Medicine
1218:at the U.S. National Library of Medicine
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109:Learn how and when to remove this message
1242:Co-Immunoprecipitation (Co-IP) Technical
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1362:Enzyme multiplied immunoassay technique
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1078:
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870:in Molecular Biology. 10.16.1–10.16.29.
514:beads. Monodisperse beads, also called
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1760:Photoactivated localization microscopy
1678:Protein–protein interaction prediction
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1024:
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743:https://doi.org/10.1074/jbc.M112448200
313:RNP immunoprecipitation (RIP and CLIP)
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349:Pull down assay using tagged proteins
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216:Chromatin immunoprecipitation (ChIP)
47:adding citations to reliable sources
18:
16:Laboratory technique in biochemistry
1635:Freeze-fracture electron microscopy
1119:
1091:Molecular & Cellular Proteomics
1013:
905:
542:
13:
1801:Protein–protein interaction assays
1312:Ouchterlony double immunodiffusion
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272:is often accomplished by applying
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1817:
1174:
1786:Biochemical separation processes
1615:Isothermal titration calorimetry
1595:Dual-polarization interferometry
145:at some point in the procedure.
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34:needs additional citations for
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503:for optimum antibody binding.
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174:Immunoprecipitation of intact
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1605:Chromatin immunoprecipitation
1302:Chromatin immunoprecipitation
1226:Chromatin+immunoprecipitation
728:
625:
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229:Chromatin immunoprecipitation
207:times for different purposes.
1796:Molecular biology techniques
1668:Protein structural alignment
1653:Protein structure prediction
1347:Chemiluminescent immunoassay
1327:Counterimmunoelectrophoresis
1200:Resources in other libraries
934:10.4049/jimmunol.147.10.3445
589:
561:
501:surface area-to-volume ratio
466:"high capacity disadvantage"
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192:protein–protein interactions
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1752:Super-resolution microscopy
1658:Protein function prediction
1586:Peptide mass fingerprinting
1581:Protein immunoprecipitation
1457:Direct fluorescent antibody
1135:The Journal of Cell Biology
696:) followed by gel staining.
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10:
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1104:10.1074/mcp.M500227-MCP200
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534:Agarose vs. magnetic beads
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1610:Surface plasmon resonance
1600:Microscale thermophoresis
1590:Protein mass spectrometry
1552:Green fluorescent protein
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1479:Total complement activity
1424:
1390:
1337:
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1195:Resources in your library
891:. Springer. p. 520.
462:"high capacity advantage"
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367:glutathione-S-transferase
363:green fluorescent protein
307:ChIP-on-chip or ChIP-chip
287:polymerase chain reaction
264:) in living cells can be
136:out of solution using an
1630:Cryo-electron microscopy
1442:Complement fixation test
1230:Medical Subject Headings
1220:Medical Subject Headings
689:(sodium dodecyl sulfate-
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224:ChIP-sequencing workflow
186:members of the complex.
148:
1663:Protein–protein docking
1576:Protein electrophoresis
885:Rosenberg, Ian (2005).
830:10.1126/science.1090095
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490:Superparamagnetic beads
1562:Protein immunostaining
1317:Radial immunodiffusion
564:section of this page.
431:Technological advances
350:
225:
128:) is the technique of
1620:X-ray crystallography
1432:Diagnostic immunology
1322:Immunoelectrophoresis
1147:10.1083/jcb.200504140
921:Journal of Immunology
775:10.1038/nprot.2006.47
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258:transcription factors
235:binding sites on the
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58:"Immunoprecipitation"
1547:Protein purification
1452:Immunohistochemistry
43:improve this article
1572:Gel electrophoresis
1447:Immunocytochemistry
1416:Latex fixation test
1294:Immunoprecipitation
1216:Immunoprecipitation
1186:Immunoprecipitation
1051:10.1038/nature06404
1043:2007Natur.450..683A
991:10.1038/nature06405
983:2007Natur.450..695A
822:2003Sci...302.1212U
816:(5648): 1212–1215.
722:secondary antibody.
694:gel electrophoresis
644:gel electrophoresis
122:Immunoprecipitation
1715:Display techniques
1567:Protein sequencing
1382:Immunofluorescence
1377:Radiobinding assay
397:or on microscopic
369:(GST) tag and the
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319:ribonucleoproteins
226:
1806:Immunologic tests
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1722:Bacterial display
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1491:
1469:Skin allergy test
1181:Library resources
977:(7170): 695–701.
898:978-0-8176-4340-9
868:Current Protocols
705:mass spectrometry
648:mass spectrometry
524:Thermo Scientific
496:superparamagnetic
392:superparamagnetic
247:or tissues. The
239:for a particular
176:protein complexes
119:
118:
111:
93:
1813:
1737:Ribosome display
1673:Protein ontology
1519:
1512:
1505:
1496:
1495:
1400:Hemagglutination
1372:Radioimmunoassay
1270:
1263:
1256:
1247:
1246:
1169:
1168:
1158:
1126:
1117:
1116:
1106:
1082:
1071:
1070:
1037:(7170): 683–94.
1026:
1011:
1010:
966:
955:
954:
936:
912:
903:
902:
882:
871:
864:
858:
857:
801:
795:
794:
760:
751:
745:
738:
716:chemiluminescent
652:western blotting
543:Binding capacity
114:
107:
103:
100:
94:
92:
51:
27:
19:
1821:
1820:
1816:
1815:
1814:
1812:
1811:
1810:
1791:Protein methods
1776:
1775:
1774:
1769:
1746:
1710:
1706:Secretion assay
1682:
1639:
1533:
1523:
1493:
1488:
1464:Epitope mapping
1420:
1386:
1333:
1307:Immunodiffusion
1288:
1274:
1206:
1205:
1204:
1189:
1188:
1184:
1177:
1172:
1127:
1120:
1097:(12): 1933–41.
1083:
1074:
1027:
1014:
967:
958:
927:(10): 3445–51.
913:
906:
899:
883:
874:
865:
861:
802:
798:
758:
752:
748:
739:
735:
731:
660:
628:
623:
614:
606:high-throughput
601:
592:
570:
545:
536:
492:
478:
438:
433:
424:
407:
388:
380:
343:
341:Tagged proteins
335:cDNA sequencing
315:
305:was also used (
295:ChIP-sequencing
218:
172:
156:
151:
115:
104:
98:
95:
52:
50:
40:
28:
17:
12:
11:
5:
1819:
1809:
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1803:
1798:
1793:
1788:
1771:
1770:
1768:
1767:
1762:
1756:
1754:
1748:
1747:
1745:
1744:
1739:
1734:
1729:
1724:
1718:
1716:
1712:
1711:
1709:
1708:
1703:
1698:
1692:
1690:
1684:
1683:
1681:
1680:
1675:
1670:
1665:
1660:
1655:
1649:
1647:
1645:Bioinformatics
1641:
1640:
1638:
1637:
1632:
1627:
1622:
1617:
1612:
1607:
1602:
1597:
1592:
1583:
1578:
1569:
1564:
1559:
1554:
1549:
1543:
1541:
1535:
1534:
1522:
1521:
1514:
1507:
1499:
1490:
1489:
1487:
1486:
1481:
1476:
1471:
1466:
1461:
1460:
1459:
1449:
1444:
1439:
1434:
1428:
1426:
1422:
1421:
1419:
1418:
1413:
1412:
1411:
1396:
1394:
1388:
1387:
1385:
1384:
1379:
1374:
1369:
1364:
1359:
1354:
1349:
1343:
1341:
1335:
1334:
1332:
1331:
1330:
1329:
1324:
1319:
1314:
1304:
1298:
1296:
1290:
1289:
1273:
1272:
1265:
1258:
1250:
1239:
1238:
1233:
1223:
1213:
1203:
1202:
1197:
1191:
1190:
1179:
1178:
1176:
1175:External links
1173:
1171:
1170:
1118:
1072:
1012:
956:
904:
897:
872:
859:
796:
746:
732:
730:
727:
726:
725:
724:
723:
708:
697:
691:polyacrylamide
681:
678:
674:
671:
667:
664:
659:
656:
627:
624:
622:
619:
613:
610:
608:applications.
600:
597:
591:
588:
569:
566:
544:
541:
535:
532:
491:
488:
477:
474:
437:
434:
432:
429:
423:
420:
406:
403:
387:
384:
379:
376:
342:
339:
314:
311:
303:DNA microarray
301:. Previously,
291:plasmid vector
217:
214:
213:
212:
208:
204:
171:
168:
155:
152:
150:
147:
117:
116:
31:
29:
22:
15:
9:
6:
4:
3:
2:
1818:
1807:
1804:
1802:
1799:
1797:
1794:
1792:
1789:
1787:
1784:
1783:
1781:
1766:
1763:
1761:
1758:
1757:
1755:
1753:
1749:
1743:
1742:Yeast display
1740:
1738:
1735:
1733:
1732:Phage display
1730:
1728:
1725:
1723:
1720:
1719:
1717:
1713:
1707:
1704:
1702:
1701:Protein assay
1699:
1697:
1694:
1693:
1691:
1689:
1685:
1679:
1676:
1674:
1671:
1669:
1666:
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1651:
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1646:
1642:
1636:
1633:
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1628:
1626:
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1618:
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1613:
1611:
1608:
1606:
1603:
1601:
1598:
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1587:
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1579:
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1573:
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1540:
1536:
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1513:
1508:
1506:
1501:
1500:
1497:
1485:
1482:
1480:
1477:
1475:
1472:
1470:
1467:
1465:
1462:
1458:
1455:
1454:
1453:
1450:
1448:
1445:
1443:
1440:
1438:
1435:
1433:
1430:
1429:
1427:
1423:
1417:
1414:
1410:
1407:
1406:
1405:
1404:Hemagglutinin
1401:
1398:
1397:
1395:
1393:
1392:Agglutination
1389:
1383:
1380:
1378:
1375:
1373:
1370:
1368:
1365:
1363:
1360:
1358:
1355:
1353:
1350:
1348:
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1336:
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1310:
1309:
1308:
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1303:
1300:
1299:
1297:
1295:
1291:
1286:
1282:
1278:
1277:Medical tests
1271:
1266:
1264:
1259:
1257:
1252:
1251:
1248:
1244:
1243:
1237:
1234:
1231:
1227:
1224:
1221:
1217:
1214:
1211:
1208:
1207:
1201:
1198:
1196:
1193:
1192:
1187:
1182:
1166:
1162:
1157:
1152:
1148:
1144:
1141:(2): 225–35.
1140:
1136:
1132:
1125:
1123:
1114:
1110:
1105:
1100:
1096:
1092:
1088:
1081:
1079:
1077:
1068:
1064:
1060:
1056:
1052:
1048:
1044:
1040:
1036:
1032:
1025:
1023:
1021:
1019:
1017:
1008:
1004:
1000:
996:
992:
988:
984:
980:
976:
972:
965:
963:
961:
952:
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940:
935:
930:
926:
922:
918:
911:
909:
900:
894:
890:
889:
881:
879:
877:
869:
863:
855:
851:
847:
843:
839:
835:
831:
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823:
819:
815:
811:
807:
800:
792:
788:
784:
780:
776:
772:
768:
764:
757:
750:
744:
737:
733:
721:
717:
713:
710:Transfer and
709:
706:
702:
698:
695:
692:
688:
685:
684:
682:
679:
675:
672:
668:
665:
662:
661:
655:
653:
649:
645:
640:
636:
632:
618:
609:
607:
596:
587:
583:
579:
576:
565:
563:
557:
553:
549:
540:
531:
529:
525:
521:
517:
513:
509:
504:
502:
497:
487:
484:
473:
471:
467:
464:can become a
463:
458:
454:
450:
446:
444:
428:
419:
415:
413:
402:
400:
396:
393:
383:
375:
372:
368:
364:
359:
358:
347:
338:
336:
332:
328:
324:
320:
310:
308:
304:
300:
296:
292:
288:
284:
280:
275:
271:
267:
263:
259:
254:
252:
251:
246:
242:
238:
234:
230:
222:
209:
205:
201:
197:
196:
195:
193:
187:
185:
181:
177:
167:
165:
161:
146:
144:
139:
135:
131:
130:precipitating
127:
123:
113:
110:
102:
91:
88:
84:
81:
77:
74:
70:
67:
63:
60: –
59:
55:
54:Find sources:
48:
44:
38:
37:
32:This article
30:
26:
21:
20:
1727:mRNA display
1696:Enzyme assay
1580:
1557:Western blot
1539:Experimental
1437:Nephelometry
1293:
1287:86000–86849)
1240:
1185:
1138:
1134:
1094:
1090:
1034:
1030:
974:
970:
924:
920:
887:
862:
813:
809:
799:
769:(1): 302–7.
766:
762:
749:
736:
712:Western blot
641:
637:
633:
629:
615:
602:
593:
584:
580:
571:
558:
554:
550:
546:
537:
512:polydisperse
508:monodisperse
505:
493:
482:
479:
465:
461:
456:
452:
448:
441:
439:
425:
416:
408:
389:
381:
355:
352:
316:
274:formaldehyde
270:crosslinking
266:cross-linked
255:
248:
227:
188:
183:
179:
173:
164:crude lysate
157:
125:
121:
120:
105:
96:
86:
79:
72:
65:
53:
41:Please help
36:verification
33:
1765:Vertico SMI
1625:Protein NMR
1409:Coombs test
1339:Immunoassay
720:fluorescent
483:preclearing
476:Preclearing
412:Protein A/G
365:(GFP) tag,
321:(RNPs). In
1780:Categories
1474:Patch test
1281:immunology
1212:at ufl.edu
763:Nat Protoc
729:References
626:Background
599:Automation
520:Invitrogen
516:microbeads
410:coated in
395:microbeads
283:sonication
132:a protein
69:newspapers
1367:RAST test
838:1095-9203
677:possible.
590:Equipment
575:sepharose
528:Millipore
470:sepharose
422:Selection
143:substrate
99:July 2008
1532:of study
1526:Proteins
1279:used in
1165:16027220
1113:16155292
1059:18046405
999:18046406
854:23420615
846:14615540
791:25925403
783:17406249
703:(MALDI)
687:SDS-PAGE
621:Protocol
405:Indirect
371:FLAG-tag
299:cistrome
262:histones
138:antibody
1530:methods
1357:ELISpot
1156:2171418
1067:2171750
1039:Bibcode
1007:4431057
979:Bibcode
951:3256313
943:1834740
818:Bibcode
810:Science
612:Summary
562:Agarose
443:agarose
436:Agarose
399:agarose
378:Methods
250:in vivo
241:protein
200:epitope
184:unknown
160:protein
134:antigen
83:scholar
1528:: key
1484:MELISA
1232:(MeSH)
1222:(MeSH)
1183:about
1163:
1153:
1111:
1065:
1057:
1031:Nature
1005:
997:
971:Nature
949:
941:
895:
852:
844:
836:
789:
781:
526:, and
386:Direct
327:RT-PCR
237:genome
85:
78:
71:
64:
56:
1688:Assay
1425:Other
1352:ELISA
1063:S2CID
1003:S2CID
947:S2CID
850:S2CID
787:S2CID
759:(PDF)
670:form.
658:Steps
445:beads
329:. In
279:lysed
245:cells
203:used.
180:known
149:Types
90:JSTOR
76:books
1161:PMID
1109:PMID
1055:PMID
995:PMID
939:PMID
893:ISBN
842:PMID
834:ISSN
779:PMID
568:Cost
510:and
449:from
357:tags
331:CLIP
260:and
62:news
1285:CPT
1151:PMC
1143:doi
1139:170
1099:doi
1047:doi
1035:450
987:doi
975:450
929:doi
925:147
826:doi
814:302
771:doi
718:or
323:RIP
309:).
233:DNA
45:by
1782::
1159:.
1149:.
1137:.
1133:.
1121:^
1107:.
1093:.
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1075:^
1061:.
1053:.
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1001:.
993:.
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959:^
945:.
937:.
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875:^
848:.
840:.
832:.
824:.
812:.
808:.
785:.
777:.
765:.
761:.
650:,
646:,
530:.
522:,
457:to
453:to
337:.
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126:IP
1588:/
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1167:.
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1009:.
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793:.
773::
767:1
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124:(
112:)
106:(
101:)
97:(
87:·
80:·
73:·
66:·
39:.
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
