Using Rabbit Antibodies for the Analysis of a Greater Epitope Range

The immune system of rabbits develops antibody-antigen affinity in a different way than the immune system of mice. 

The differences in rabbit immune system development are mostly thought to be the result of the larger bank of B-cells and the reduced immune dominance of rabbits. The benefit of rabbit immune development is that it provides wider epitope recognition during development.

An example of this, where rabbit and murine immune responses are compared, can be seen below. The example demonstrates that rabbit antisera can recognize a greater number of epitopes than the mouse equivalent when using a western blot method.

Abcam’s experience in producing antibodies has shown that the immune system of rabbits tends to produce a greater variety of antibodies, which can recognize distinctive epitopes.

RabMAb vs Mouse Monoclonal Comparison

In addition a great number of clinically significant human antigen proteins are highly similar in both humans and mice. As a result these antigens usually are less immunogenic if a mouse or rat is used as the host.

Rabbits, with their unique immune system, provide greater diversity and enhanced affinity maturation. RabMAb primary antibodies can target a greater range of epitopes, facilitating the identification and analysis of novel antigens.

Case study: Thiophosphate Ester RabMAb Primary Antibody [51-8] (ab92570)

The phosphorylation of proteins is one of the most heavily studied signaling mechanisms known, occurring in both prokaryotic and eukaryotic organisms. Phosphorylation, where a kinase adds a phosphate unit (PO4) to a protein, forms the backbone of cell signaling pathways, in addition to modulating protein properties (via structure-function relationships) that regulate all basic cellular processes.1,2,3

Despite this, the biochemical analysis of substrates that associate with a specific kinase of interest (KOI) is both difficult and limited. This is because most phosphoproteins are present in very low concentrations, and there is also weak binding and low substoichiometry phosphorylation of the kinase-to-substrate interaction.1,4

To solve these problems a new method of bio-orthogonal affinity purification and identification of direct kinase substrates has been developed.6

Following this method KOI first accepts ATPγS, following which it can thiophosphorylate its direct substrates. Secondly, the substrates thiophospholyated site is then alkylated using p-nitrobenzylthiophosphate (PNBM). Following alkylation a thiophosphate ester, that is selective towards the rabbit monoclonal antibody (clone 51-8), is used to analyze the tagged substrates (A Nature methods 2007 paper contains a diagram of this method).

This novel method of semi-synthetic epitope tagging gives researchers a new method of characterizing and isolating the substrates of different kinases.4,5,6

Using a unique RabMAb primary antibody kinase substrates that contain thiophosphate esters (following the kinase reaction) can be detected. This method is described by J. Allen et al. in Nature methods 2007 (see below figure).

Using an RabMAb primary antibody gives researchers the ability to follow this method and study the interesting relationships between kinases and their substrates.

The antibody also provides the benefit of being context independent, being able to recognize tyrosine, threonine and serine thiophosphorylated residues in a wide range of different kinase phosphorylation structures.

References

  1. Johnson, S.A. and Hunter, T. Nature Methods 2, 17–25, 2005
  2. Hunter, T. Signaling−2000 and beyond. Cell 100, 113−127, 2000
  3. Cozzon, A.J. Ann. Rev. Microbiol. 42, 97–125, 1988
  4. Cai, D, Article Review, Mol. BioSyst., 3, 516 – 517, 2007
  5. Allen, J.J. et al. J. Am. Chem. Soc., 127(15), 5288–5289, 2005
  6. Allen, J.J. et al. Nature Methods, 4(6), 511–516, 2007. (PubMed)

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: May 6, 2019 at 5:13 AM

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