Agarose Beads for Immunoprecipitation and Antibody Purification

Protein A and Protein G Plus Agarose Beads

Protein A Agarose binds to the Fc region of IgG from a variety of species. Can be used to purify classes, subclasses, and fragments of immunoglobulins as well as for isolation of immune complexes. Reusable resin contains 10 mg protein A/mL of beaded agarose matrix. Binding capacity: ≥40 mg human IgG/mL resin.

Protein G Agarose binds to the Fc portion of immnuoglobulins (Igs) from various species; useful for binding to Igs that do not react well with protein A. Can be used for antibody immunoprecipitation procedure and to purify immunoglobulins and IgG fractions.

Protein A and Protein G Plus are proteins of microbial origin that bind specifically to mammalian immunoglobulins. When coupled to agarose, these reagents provide efficient purification and immunoprecipitation of antibodies. Immunoglobulins of various species interact differently with the two proteins, and the combination of Protein G Plus/Protein A can be used to provide the characteristics of each in one reagent. Note that Protein G Plus differs from the standard Protein G by lacking albumin binding and protease cleavage sites. Two forms of agarose preparations are available; one is designed for purification in column or batch mode, and the other is provided as a ready-to-use suspension containing BSA to reduce non-specific adsorption and is designed for immunoprecipitation applications. Both use highly purified recombinant proteins, cross-linked agarose, and stable covalent coupling chemistry.

Protein A, Protein G Plus, and Protein G Plus/Protein A Agarose Suspensions are provided ready-to-use as slurries containing 30% agarose conjugate by volume. Each contains BSA as a blocking agent and binds 20 mg IgG/ml packed beads under static conditions. These suspensions should be used for precipitation of primary and secondary antibodies and are not intended for purification.

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Montage® Antibody Purification Kits

Montage® Antibody Purification Kits are designed for fast and easy antibody purification from serum, ascites or cell culture supernatants. The kits include spin columns pre-packed with high capacity PROSEP-A or G media. There are no pumps to setup and no columns to pack. All you need is a centrifuge to purify 10–20 mg in less than 60 minutes.

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The protocol is easy to follow and, unlike traditional gravity flow methods, the use of centrifugal purification allows for walk-away convenience. Results show that the spin columns can be regenerated up to 10X with no loss of binding capacity or reduced specificity.

Human IgG was purified 10 times from normal serum using a regenerated PROSEP-G spin column.

Figure 1: Human IgG was purified 10 times from normal serum using a regenerated PROSEP-G spin column.

Advantages

  • Montage® kits offer the researcher a total antibody purification process from the initial clarification stage to the final antibody concentration step.
  • Montage® kits use a spin column format designed to eliminate tedious chromatographic steps and expensive hardware normally associated with Protein A chromatography.
  • PROSEP-A media has a proven track record for biopharmaceutical therapeutic antibody production.
  • Minimal hold-up volume of the Protein A resin plug ensures high antibody recovery.
  • The reusable spin columns are sufficient for 10 purifications each and multiple samples can be processed simultaneously.
  • The beaded supports offer excellent flow properties that, combined with the tapering of the spin column, provide uniform flow paths enabling optimal use of the media bed in swinging bucket rotors.

Binding Affinities of Protein A and Protein G

Antibody Protein A Protein G Antibody Protein A Protein G
Human lgG1 ++ ++ Rat lgM +/-- --
Human lgG2 ++ ++ Rabbit lgG ++ ++
Human lgG3 -- ++ Hamster lgG + ++
Human lgG4 ++ ++ Guinea Pig lgG ++ +
Human lgA + -- Bovine lgG + +
Human lgD + -- Sheep lgG +/-- +
Human lgE + -- Goat lgG +/-- +
Human lgM + -- Pig lgG ++ ++
Mouse lgG1 + + Chicken lgG -- +/--
Mouse lgG2a ++ ++ Fragments
Mouse lgG2b ++ ++ Human Fab + +
Mouse lgG3 + ++ Human F(ab)2 + +/--
Mouse lgM +/-- -- Human ScFv + --
Rat lgG ++ ++ Human Fc + --
Rat lgG1 +/-- + Human K -- --
Rat lgG2a +/-- ++ Human -- +
Rat lgG2b +/-- +  

Legend: ++ = Strong affinity; + = Moderate/slight affinity; 
+/-- = Requires evaluation; -- = No affinity

Materials