Polyclonal Antibody Purification

Why Polyclonal Antibody Need Purification

For purification of polyclonal antibodies, protein A/G purification is the purification method of choice, which involves purifying antibodies from animal serum. Although protein A/G purified polyclonal antibody can be good enough for some research applications, it may not be suitable in many other situations, probably due to low detection sensitivity or unacceptable background. In many cases, further purification and fractionation of the antigen specific antibodies from the total IgG antibodies can increase detection sensitivity and reduce undesirable background. This can be achieved by purifying with antigen conjugated resin.

Sino Biological has developed in-house expertise in conjugating proteins and antigens to agarose resin, and has gained sufficient hands-on experience in purifying high-quality antigen-specific polyclonal antibodies. Our custom polyclonal antibody services have been highly reliable and cost-attractive to customers.

How to Purify Antibody from Serum

When antiserum has been collected for purification, ammonium sulfate precipitation is performed. This removes many sticky proteins from the antiserum and allows for a cleaner preparation of material to be subjected to the affinity column. Different purification methods are often used in combination to purify a better polyclonal antibody from serum, which is critical to the performance of the intended applications. View more about "Antibody Purification Methods".

Protein A/G purification

Because antibodies have predictable structure, including relatively invariant domains, it has been possible to identify certain protein ligands that are capable of binding generally to antibodies, regardless of the antibody's specificity to antigen. Protein A and Protein G are bacterial proteins whose antibody-binding properties have been well characterized.These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species.

Protein A/G purification is a quick purification method for polyclonal antibodies that have been generated against recombinant proteins or antigens. Due to their ability to bind the constant (Fc) region of IgG from various species, serum proteins as well as IgM antibodies can be removed. It should be noted that the final preparation will contain total IgG antibodies and specific antibodies.

Sino Biological has manufactured Protein A and Protein G Agarose Beads / resins, which can be used to purify antibody from multiple sources, such as mouse IgG2a/2b/3, rat IgG2c, human IgG1/2/4 and rabbit IgG. Both Protein A and Protein G Agarose Beads have high binding capacity.

Antibody purification with protien A/G
Fig 1. Protien A/G affinity antibody chromatography

Antigen affinity purification

After protein A/G purification, antigen affinity purification is another type of affinity purification and results in the purest polyclonal antibodies with the least amount of cross-reactivity. Antigen-specific purified antibodies exhibit the highest specificity and sensitivity that can be obtained from serum. Many applications, such as IHC and IF, benefit from this process as polyclonal antibodies can be used at lower dilutions without increasing signal background.

Antigen affinity purification can be performed by immobilizing target-specific antibodies to the resin in order to purify a polyclonal antibody. After antiserum is incubated in the column, antigen-specific antibodies are purified by pH gradient elution. Purified antibodies are then concentrated and final concentration should be measured. Antibody titer is also performed by ELISA.

Sino Biological has accumulated an extensive set of experience and know-how in affinity purification using protein A/G resins or immunogen affinity columns to increase antibody specificity and to reduce background binding.

Antigen affinity antibody purification
Fig 2. Antigen affinity antibody chromatography

References

1. Hober, S., Nord, K., & Linhult, M. (2007). Protein A chromatography for antibody purification. Journal of Chromatography B, 848(1), 40-47.
2. Low, D., O'Leary, R., & Pujar, N. S. (2007). Future of antibody purification. Journal of Chromatography B, 848(1), 48-63.
3. Fishman, J. B., & Berg, E. A. (2019). Antibody Purification and Storage. Cold Spring Harbor Protocols, 2019(5), pdb-top099101.

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