Immunoprecipitation practical tools magnetic beads and agarose resin performance competition
IP experiments are the choice of proteomics research partners when we need to concentrate proteins in small amounts and perform a single assay.
Co-IP is almost mandatory when we need to demonstrate protein-protein interactions. Using cell lysis samples, the interaction of intracellular proteins was proved and the molecular mechanism of the phenomenon was elucidated.
At this time, the practical tool for IP experiment is the most urgent problem to be solved by the majority of proteomics researchers. Then how to choose the most suitable IP tool for their primary antibody? Here is a comparison of IP utilities (magnetic beads and agarose resin) :
First, because IP was developed as an improved method for column affinity chromatography, it was initially done using small amounts (10 -- 25 µL) of agarose resin in microcentrifuge tubes. Agarose is a spongy structure of various shapes and sizes (50-150 μm in diameter). Resin must be separated from the sample and buffer by centrifugation: the clear resin can be precipitated and the solution carefully removed, or the resin can be retained using a microcentrifuge filter and the solution collected in the centrifuged tube. These limits limit how scalable or automated agarose IP can be.
In recent years, magnetic particles have largely replaced agarose as the preferred support for immunoprecipitation and microaffinity purification methods. Magnetic particles are spherical solids and antibody binding is limited to the surface of each bead. Although magnetic beads do not have the advantage of a porous center to increase binding capacity, they are significantly smaller than agarose microbeads (1-4 μm in diameter) and can provide a large enough total surface area for high volume antibody binding.
To understand their performance differences, start with their microscopic morphology:
The spongy structure of the agarbose bead, shown here, is 50-150 μm in diameter and binds antibodies (and thus target proteins) directly, efficiently and rapidly, without the need for specialized equipment.
The structure of the magnetic bead, shown here, is solid and antibody binding is limited to the surface of the bead. The magnetic beads have a diameter of 1-4 μm, which is significantly smaller than that of agarose beads. Although the magnetic beads have no porous center to increase the binding capacity, the number of magnetic beads per volume is more than that of agarose beads, so that the magnetic beads have enough antibody binding surface area to meet the high capacity of antibody binding.
In order to give you a better understanding of magnetic beads and agarose resin, we have compared a number of their data, as shown in the following table:
|performance||Capacity||Yield||Reproducibility||Purity-Specificity||Easy of Use||Speed||Multiple Samples||Automation|
The functional comparison between immunoprecipitated magnetic beads and agarose resins is shown below:
|pros and cons||Agarose Beads||Magnetic Beads|
|Capacity||Porous (spongy), it has a large surface-volume ratio, high antibody binding ability.||The outer surface is smooth and porous, and the theoretical binding ability of magnetic beads is lower than that of agarose.|
|Yield||Antibodies fixed to spongy agarose do not always bind to the target protein (usually large protein complexes) in the sample, and antibodies may be lost during the washing step (centrifugation)||Antibodies bound to the surface of the magnetic bead can bind to the antigen, and antibodies are rarely lost in the mild washing step (magnet) at an equal or higher rate.|
|Reproducibility and Purity-Specificity||It is difficult to completely remove the buffer without damaging and approaching some of the precipitated resins, and agarose usually requires long incubation times (allowing diffusion of the solution into internal space) and pre-purification steps (controlling nonspecific binding of non-target proteins).||All magnetic beads are fixed on the wall of the tube, so the buffer can be removed without touching the precipitation of magnetic beads. The size is more uniform, and prepurification steps are generally not required. The reproducibility and purity of magnetic beads are generally higher than that of agarose.|
|Easy, speed and Automation||IP requires a long incubation time, pre-purification steps and multiple centrifuges, so it requires a lot of manual handling, with a total time of 1-1.5 hours.||A single magnetic bead immunoprecipitation can be used in about 30 minutes and multiple steps can be performed automatically.|
The comparison is summarized as follows:
Magnetic beads: Immunoprecipitation is performed using magnetic beads (i.e., when sample volume < 2 mL). Magnetic beads can achieve a balance of binding power/yield, repeatability, purity and cost savings in routine small-scale isolation of specific proteins and protein complexes. Magnetic beads are the best choice when manual and automated standard IP, co-IP, ChIP, chip-seq, RIP and pull-down reactions are performed and immediately used for subsequent detection analysis.
Agarose resin: Protein purification using agarose resin (i.e., when sample volume > 2 mL). Agarose is best suited for column affinity chromatography or stand-alone large rotating cup immunoprecipitation reactions when large volumes of antibodies are cost-effective and large volumes of target proteins are purified for multiple downstream analyses.
Related products recommendation
|Product NO.||Product Name||Application|
|KTD104-EN||Universal IP/Co-IP Toolkit (Magnetic Beads)||IP/Co-IP|
|KTD105-EN||Universal IP/Co-IP Toolkit (Agarose)||IP/Co-IP|
|KTP2070||PurKine™ Antibody Purification Kit (Protein A/G)||Protein Purification|
|BMR2070||PurKine™ Protein A/G Resin 4FF||Protein Purification|
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