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The motivating question was experimental: how could one recover the order in a protein when breaking all its peptide bonds destroys that order? Frederick Sanger and colleagues used complementary tests on portions of insulin. The following is an explanation of their evidence, not a laboratory recipe.

First, mark ends before destroying the chains. Sanger used the colored reagent fluorodinitrobenzene, abbreviated FDNB, to label free amino groups. After acid hydrolysis, the attached label remained on the end-residue products. Separating these derivatives by their different movement through solvents and comparing them with reference derivatives made the labels readable. The N-terminus products identified glycine and phenylalanine. Sanger emphasized that finding a stable label and a way to separate its products were both essential. See Sanger, Citation Classic (1985), discussing The free amino groups of insulin (1945). FDNB also labels suitable free amino groups on amino acid side chains, so a labeled product is not automatically an N-terminus product. See Sanger, The Chemistry of Insulin, Nobel lecture (1958), pp. 546–547.

Next, separate chains and preserve local order. Oxidation with performic acid broke the protein disulfide bonds, allowing A and B chains to be isolated. Partial hydrolysis and enzyme treatments produced fragments; these were separated and their composition and local sequences characterized. Overlaps between different fragment sets linked smaller sequences into longer ones. Many more fragments than minimally necessary were checked for consistency. See Sanger's authored Nobel lecture, pp. 547–552: chain separation, fragment analysis and sequence reconstruction.

The toy fragments make the logic visible. Suppose one cut pattern yields MAG and AMG, and another yields MA, GA and MG. The first set alone permits MAGAMG or AMGMAG. The fragment GA in the second set crosses the MAG–AMG boundary in MAGAMG; it does not occur in AMGMAG. Among concatenations of the first set, therefore, the second set selects MAGAMG. This example assumes intact, correctly identified fragments from one homogeneous chain and known fragment membership in each cut pattern. Repeated stretches, missing fragments or sample mixtures can leave ambiguities; the picture illustrates inference from overlaps, not a universal guarantee of reconstruction.

Finally, determine the protein disulfide bonds separately. Oxidation used to separate whole chains erases their original connections. Sanger and colleagues instead studied fragments still joined by protein disulfide bonds, then oxidized the isolated joined fragments to identify their component pieces and locate the connected chain regions. An important trap was rearrangement of disulfide bonds during treatment: products can acquire connections absent from the starting protein. Their work therefore had to establish conditions that prevented rearrangement before interpreting a linked fragment as evidence. See Ryle and Sanger, A Disulphide Interchange Reaction, Nature 171, 1025–1026 (1953) and Sanger's Nobel lecture, pp. 553–554: preserving and identifying disulfide connections. The result was an experimentally constrained chemical structure rather than a composition list or a photograph of a fold.

A storage compartment that participates in regulated exocytosis; the insulin example distinguishes storage of a hormone from storage of glycogen fuel.

This deliberately synthetic differential equation model illustrates response direction and lag. Every plotted number is chosen for teaching; none is a biological measurement or a fitted human parameter. Let externally prescribed, dimensionless $g>0$ represent a glucose stimulus relative to a reference. Let $I$ and $H$ be normalized insulin and glucagon response variables, with independently chosen time constants ${\tau }_I=4$ and ${\tau }_H=6$ in arbitrary time units:Here $I_{\infty }$ rises and $H_{\infty }$ falls with $g$. Their targets add to one by construction; the evolving $I+H$ need not equal one because their time constants differ. Start with $g=1$ and $I=H=1/2$. Set $g=1.6$ from $t=10$ to $40$, $g=0.65$ from $t=40$ to $70$, and $g=1$ otherwise. Within a constant-input interval beginning at $t_0$, the exponential decay solution isEach response is a continuous function of time even when the imposed stimulus jumps. The chosen starting values stay between zero and one, since each solution is a weighted arithmetic mean of its interval's starting value and target.

For an illustrative downstream comparison, define release tendency $R=0.3+0.6H/(1+I)$ and uptake tendency $U=(0.25+0.5I)g$, in the same arbitrary flux units. At the reference target, $R=U=0.5$. At the high-stimulus targets, $R\approx 0.343$ and $U\approx 1.094$; at the low-stimulus targets, $R\approx 0.742$ and $U\approx 0.212$. These chosen functions encode greater uptake with insulin and greater release with glucagon, moderated by insulin; they are not established enzyme kinetics. The $g$ trace is imposed externally, so the figure does not demonstrate self-correction of blood glucose. A closed model would also need a conservation of mass equation, nutrient input, finite glycogen stores, and additional tissue interactions. The qualitative restoring loops are shown separately in the insulin circuit.