• 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.

  • 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.

  • [5] 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 advantage of this technology is a very high potential binding capacity, as virtually the entire sponge-like structure of the agarose particle (50 to 150μm 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.

  • Summary[edit] 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 interest and the IP method used.

  • 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 added.

  • Alternatively, when one wants to find where the protein binds on a genome-wide scale, ChIP-sequencing 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 cistrome.

  • Because antibodies can be a cost-limiting factor, it is best to calculate backward from the amount of protein that needs to be captured (depending upon the analysis to be
    performed downstream), to 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 to the quantity of agarose that is
    needed to bind that particular quantity of antibody.

  • 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.

  • In most cases, preclearing the lysate at the start of each immunoprecipitation experiment (see step 2 in the “protocol” section below)[6] 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.

  • 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 saturated beads are used.

  • This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

  • Spin columns contain a filter that allows all IP components except the beads to flow through using a brief centrifugation and therefore provide a method to use significantly
    less agarose beads with minimal loss.

  • 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 used.

  • 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.

  • Others may argue for the use of magnetic beads because of the greater quantity of antibody required to saturate the total binding capacity of agarose beads, which would obviously
    be an economical disadvantage of using agarose.

  • Cost[edit] The price of using either type of support is a key determining factor in using agarose or magnetic beads for immunoprecipitation applications.

  • Binding capacity[edit] 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.

  • The indirect method is also used when the binding kinetics of the antibody to the protein is slow for a variety of reasons.

  • Tagged proteins[edit] Pull down assay using tagged proteins One of the major technical hurdles with immunoprecipitation is the great difficulty in generating an antibody that
    specifically targets a single known protein.

  • [5] Claims have also been made that magnetic beads are better for immunoprecipitating extremely large protein complexes because of the complete lack of an upper size limit
    for such complexes,[7][8][10] although there is no unbiased evidence stating this claim.

  • The nature of magnetic bead technology does result in less sample handling[8] 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.

  • 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.

  • 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.

  • • Repeating the experiment by targeting different members of the protein complex allows the researcher to double-check the result.

  • Types Individual protein immunoprecipitation (IP)[edit] Involves using an antibody that is specific for a known protein to isolate that particular protein out of a solution
    containing many different proteins.

  • Both methods give the same end-result with the protein or protein complexes bound to the antibodies which themselves are immobilized onto the beads.

  • 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.

  • So the decision to saturate any type of support depends on the amount of protein required, as described above in the Agarose section of this page.

  • 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.

  • 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.

  • While the magnetic capture equipment may be cost-prohibitive, the rapid completion of 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.

  • 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.

  • 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.

  • In CLIP, 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 cDNA sequencing.

  • The crosslinking is often accomplished by applying formaldehyde 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).

  • 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 50μl beads per IP).

  • 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.

  • Direct[edit] Antibodies that are specific for a particular protein (or group of proteins) are immobilized on a solid-phase substrate such as superparamagnetic microbeads or
    on microscopic agarose (non-magnetic) beads.

  • This step may be performed in a standard microcentrifuge tube, but for faster separation, greater consistency 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.

  • Selection[edit] 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.

  • 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.

  • [4][5] Preclearing[edit] 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).

  • The advantages with using tagged proteins are so great that this technique has become commonplace for all types of immunoprecipitation including all of the types of IP detailed

  • In cases where antibody saturation is not required, this technology is unmatched in its ability to capture extremely large quantities of captured target proteins.

  • Conversely, spin columns may be employed instead of normal microfuge tubes to significantly reduce the amount of agarose beads required per reaction.

  • 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 times
    for different purposes.

  • 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 beads, also called microbeads, exhibit exact uniformity, and therefore all beads exhibit identical physical characteristics, including the binding capacity and
    the level of attraction to magnets.

  • By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex.

  • 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.

  • Protocol Background[edit] 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.

  • 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.


Works Cited

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Keene JD, Komisarow JM, Friedersdorf MB (2006). “RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts”. Nat Protoc. 1 (1): 302–7. doi:10.1038/nprot.2006.47. PMID 17406249.
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6. ^ Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaz; Darnell, Robert B. (2003-11-14). “CLIP identifies Nova-regulated RNA networks in the brain”. Science. 302 (5648): 1212–1215. Bibcode:2003Sci…302.1212U.
doi:10.1126/science.1090095. ISSN 1095-9203. PMID 14615540. S2CID 23420615.
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the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI”. Journal of Immunology. 147 (10): 3445–51. doi:10.4049/jimmunol.147.10.3445. PMID 1834740. S2CID 3256313.
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LM, et al. (November 2007). “The molecular architecture of the nuclear pore complex”. Nature. 450 (7170): 695–701. Bibcode:2007Natur.450..695A. doi:10.1038/nature06405. PMID 18046406. S2CID 4431057.
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