How much peptide do you need?
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As in ordering any reagent for an experiment, a scientist needs to estimate his/her requirements for peptides and look at the costs involved before making a decision on whether an experiment using peptides is financially feasible (within budget). This article is intended to help you estimate your requirements and determine the feasibility of using peptides in your experiments.
How is the Peptide to be used?
The uses of peptides can be broadly divided into two categories: those uses where the experiment simply looks at binding between another substance and the peptide, and those where the peptide is committed to a biological system in order to look for a "biological" effect. The "biological" effect may be brought about after modification of the peptide by the biological system, and the effect observed may even be an indirect consequence of the response of the biological system to the peptide. An example of the former is a simple ELISA on solid-phase-bound peptide; an example of the latter is the use of peptide in bioassays of hormone agonist activity. The calculation of peptide requirement is much more straightforward in the first category of use, since the peptide should obey simple laws of mass action, whereas in the second category it may be necessary to establish a dose- response curve de novo for the biological system under investigation. Peptide binding systems
1. Permanently bound peptide
The most likely desired application is the determination of direct binding (e.g. of a macromolecule such as a protein) to peptide on a solid phase. The use of peptide on the same solid phase on which it was synthesized [1,2] was a breakthrough in terms of the opportunity to test many peptides and variants for antibody binding. In this case, it is possible to re-use the peptide up to 50 times by removing the bound entity [3]. The amount of peptide required on the solid phase can be vanishingly small (say, 1nmol or less). At issue here, in terms of quantity, is not the amount on each solid support (provided there is EXCESS peptide over that required for saturation binding), but rather the number of separate supports (e.g. pins) required for the experiments envisaged. In general, it would be wise to have at least a duplicate (pair) of pins of each peptide as a check on reproducibility and to allow faster generation of results when screening many samples on the same peptides (pins).
2. Bound peptide prepared from peptide solutions
When short peptides are prepared in soluble form, some means of reliable capture of the peptide onto a solid phase is required for use in a direct ELISA. Passive absorption onto plastic is unreliable [4] and inappropriate for peptides of less than about 8 residues. We advocate the use of biotinylated peptides with avidin or streptavidin-coated plates (see Pinnacles Vol.1 No.2, page 3). For assays of antibody binding to such captured peptides, in excess of 10,000 wells can be coated to saturation with 1micromole of peptide, the average yield from a single pin.
Alternatively, biotinylated peptide can be bound to avidin or streptavidin pre-bound on biotin-beads (e.g. Sigma Cat. No. B- 3272) for use as a solid phase. One micromole of biotinylated peptide, the average yield from a single pin, is sufficient to load approximately 3mL of avidin-saturated biotin-gel (biotin- binding capacity of 0.35 micromole per mL).
3. Solution phase peptide used in competition tests
Here the readout detects the complex between a macromolecular binding entity (e.g an antibody) and a ligand, and peptide is used to compete with the ligand for binding. The amount of peptide which may be required is large, particularly if the sensitivity of the detection method for the macromolecule- ligand complex is poor and the affinity of the macromolecule for the peptide is lower than for the ligand. Typically, peptide from a single pin gives about one mL of peptide solution at a concentration of 1mg/mL, which should be enough to do a competition test over the entire range of practical concentrations for one macromolecule/ligand system.
Bioassay systems using peptides
The systems with which we are most familiar are bioassays with cells of the immune system, and bioassays for peptide hormone action.
1. Bioassays with cells of the immune system
Antigenic determinants recognised by T lymphocytes (T cells) can be identified using synthetic peptides added to cultures of T cells, either unselected (e.g. peripheral blood mononuclear cells, PBMC) or T cell lines, clones or hybridomas. Peptide added to a concentration of 1 micromolar is generally sufficient for determinant identification. For microplate cultures, the peptide from a single pin is sufficient for up to 5000 wells at 1 micromolar (0.2 mL/well). Alternatively, for titration of response versus concentration (e.g. with a T cell clone), one may wish to begin with a concentration as high as 0.1mM. Up to 10mL of peptide solution at this high concentration can be made with the peptide from a single pin.
2. Bioassays with whole animals
A study of the whole animal response to analogs of the hormone angiotensin II was carried out using bolus injections of up to 3 nmol of peptide into anesthetised rats [5]. Sufficient peptide is obtained from a single pin to allow 300 such injections to be carried out.
3. Bioassays on tissue preparations
As an example, the "second messenger" response of neural tissue can be studied in slices of tissue in 500 microlitre of Ringer's saline [6] by addition of a tachykinin such as substance P, a peptide of 11 residues. Agonist is added at 1micromolar, requiring 0.5nmol per test. Thus, approximately 2000 assays can be carried out with the peptide from a single pin. Naturally, fewer assays can be performed on peptides for evaluation as antagonists if they are tested at high multiples of the agonist concentration, but conversely peptides with high activity as agonists can be used for many more assays.
Summary
While the amount of peptide needed for a research project is naturally a function of the type of assay in which it is to be used, there are many instances, particularly in the earlier investigative stages of a project, when only a small amount of peptide is required. The above examples show that sufficient peptide may be available from a single pin to complete the evaluation of a peptide prior to committing to larger scale work. Synthesis of the same peptide on multiple pins also allows economical scale-up, by a factor of 10 or more, before alternative methods of solid phase peptide synthesis become cost-competitive.
References
1. Smith, J.A., Hurrell, J.G.R., Leach, S.J. (1977). A novel method for delineating antigenic determinants: peptide synthesis and radioimmunoassay using the same solid support. Immunochemistry 14; 565-568.
2. Geysen, H.M., Meloen, R.H. and Barteling, S.J. (1984). Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA, 81; 3998-4002.
3. Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G. and Schoofs, P.G. (1987). Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102; 259-274.
4. Geerligs, H.J., Weijer, W.J., Bloemhoff, W., Welling, G.W. and Welling-Wester, S. (1988). The influence of pH and ionic strength on the coating of peptides of herpes simplex type I in an enzyme-linked immunosorbent assay. J. Immunol. Meth. 106; 239-244.
5. Rodda, S.J., Geysen, H.M. and Mason, T.J. (1991). Epitope screening and analysis using a multiple peptide synthesis system and its potential use in hormone analysis in Neuroendocrine Research Methods (ed B. Greenstein, Harwood, Chur). Chapter 44, pp 987-1004.
6. Mantyh, P.W., Vigna, S.R. and Maggio, J.E. (1991) Assays for substance P and tachykinin receptors in Methods in Neurosciences Vol. 5 (Academic, N.Y.) p404-425