…or how to make your protein attractive
Biomagnetic separation is a powerful lab tool in protein biochemistry which can be used in a wide range of applications including analysis tools (for protein solubility and protein-protein interaction studies, and for testing several purification buffers…), high throughput screening (HTS) and larger protein purification scale.
Indeed, it’s an easy, quick and efficient method to purify recombinant proteins and antibodies. Note that it also can be used to remove endotoxins and abundant protein contaminants from samples. Let’s take a look together at the large number of possibilities available and how we can help you to design the most adapted protocol for your study.
During the last few years, magnetic beads have become popular lab tools. Several suppliers even call them the better way to do protein immunoprecipitation (see Fig. 1). In some cases, Magnetic beads are certainly a little bit more expensive than classical beads. Nevertheless, there are a lot of advantages:
- Simplicity: a single step high-throughput procedure
- Ease of use: No column and centrifugation steps are needed compared to classical beads, with pipetting made easier (magnetic beads are magnetized to the side of the tube wall). Beads can be easily reused several times.
- Speed of use: time devoted to the manipulation is short (magnetize in less than 10s),
- Efficiency: washing is more stringent (with less washing steps) for higher protein purification yields and reproducibility (high performance)
Taking into account all these advantages, magnetic beads are much more attractive than classical beads. Manufacturers propose a large choice of magnetic beads with a wide range of applications making their choice sometimes quite delicate or at least tricky.
Below, I have focused on 4 of their most popular applications in recombinant protein purification which can permit you to choose easily the right beads for the right application.
Application #1 – Tagged recombinant protein purification
His-tag is the most widely used affinity tag for protein purification. A wide range of different his affinity beads are available with different metal affinity beads combined with Nickel, Cobalt, Copper, Iron, Zinc, Gallium, Aluminium, and also rarely Zirconium. The protein elution can be done in several steps by increasing progressively the imidazole concentration or in one step with high concentrations of imidazole (1 M-500 mM).
Tag affinity beads also include resins with immuno-affinity which are combined with anti-GST, anti-Flag, anti-c-myc, anti-HA or anti-V5 tag antibodies. The elution buffer must be composed of ligands with a higher affinity for the resin than the protein of interest has (for ex: glutathione, peptides, glycine…) or with high pH buffer.
Application #2 – Untagged protein / antibody purification
3 strategies are employed to purify protein without a tag:
- Magnetic beads combined with native Protein A or Protein G are used to simplify the enrichment of antibodies in the sample. Proteins A and G are bacterial proteins from Staphylococcus aureus and Streptococcus species respectively. They have a high affinity for the Fc region of IgG antibodies from a broad variety of species. The elution of the antibody can be done by using, for example, an acidic buffer (pH 2-3) or a buffer containing urea. Then, you need to use a neutralizing or an exchange buffer (by using a desalting column or a dialysis cassette).
- Custom magnetic matrix consists in coupling a custom protein to the matrix. The principle is based on specific protein-protein interactions. This technology permits to purify and to co-immunoprecipitate easily two proteins. Note that the “custom protein” used here needs to have a high affinity for the protein of interest and to be previously identify to consider this strategy.
- The Streptavidin Magnetic Beads can be used in order to isolate biotinylated molecules. The interaction between streptavidin and biotin is formed very rapidly. Interestingly, this interaction is unaffected by wide extremes of temperature, organic solvents, pH and other denaturing agents. There is a large variety of biotinylation reagents that target different functional groups like side chains of primary amines, sulfhydryls, carboxyls, carbohydrates, tyrosine and histidine, and also bases (guanidine and cytosine). There are many biotinylation kits that enable simple and efficient biotin labeling of antibodies and proteins. You just need to choose the optimal biotinylation reagent for your experiments. During the purification, the impurities are removed by washing, and the protein of interest can be eluted using a buffer containing biotin.
Application #3 – Endotoxin Removal
Some beads have been specifically designed for removing endotoxin which are contaminants produced by gram-negative bacteria and which can induce an inflammation when injected in mammals/humans. Polymixin B immobilized on beads permits to eliminate easily and efficiency endotoxin from your samples. Endotoxin LAL tests which are FDA approved are required to verify the entire elimination of endotoxin before injection (take a look at Endotoxin level measurement).
Application #4 – Abundant protein removal like IgG, IgA, BSA, Complement C3
Affinity depletion of abundant proteins from human plasma is useful in proteomic studies. Each supplier possesses beads against specific plasma proteins (that are meant to be depleted). You need to choose which plasma proteins are the worst contaminants in your experimental sample in order to choose the most appropriate resin. Then, you just need to incubate your sample with beads which are able to capture selected contaminants.
Magnetic beads are essential lab tools to be used not only to purify recombinant proteins but also DNA and RNA with a high throughput procedure and with the capacity to discriminate mutations. They are also the simplest method for the activation and the expansion of T-cells.
But that’s another story…
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