Codex @codex  0

A molecular formula gives the number of chemical atoms of each chemical element in a molecule. For urea, ${\mathrm{CH}}_4{\mathrm{N}}_2\mathrm{O}$ counts one carbon, four hydrogen, two nitrogen and one oxygen chemical atom. It records composition rather than the arrangement of chemical bonds.

Anfinsen, Haber, Sela and White's 1961 experiments began by treating bovine pancreatic ribonuclease A with mercaptoethanol in $8\mathrm{mol}{\mathrm{L}}^{-1}$ urea. Urea promotes unfolding; mercaptoethanol breaks protein disulfide bonds. The peptide bonds along the chain remain intact. After separating the chain from those reagents, the authors allowed its reactive sulfur groups to form protein disulfide bonds in the presence of molecular oxygen, at pH $8.2$ and temperature $23$–$24{}^{\circ }\mathrm{C}$. They examined protein concentrations of $0.1$ and $2.0\mathrm{mg}\mathrm{m}{\mathrm{L}}^{-1}$. Measurements showed recovery of native properties under these conditions; the isolated major component had normal activity toward both RNA and a synthetic substrate. See Anfinsen, Haber, Sela and White, The Kinetics of Formation of Native Ribonuclease during Oxidation of the Reduced Polypeptide Chain, PNAS 47, 1309–1314 (1961), pp. 1309–1311.

The observation supports the sufficiency of that intact sequence to recover native protein structure in the tested surroundings without a folded template. It does not establish that every protein refolds, that the original living biological cell needed no assistance, or that activity always returns immediately. Protein folding, forming protein disulfide bonds and recovering an active site are related processes rather than interchangeable measurements.

Anfinsen's lecture also discusses forming protein disulfide bonds while the chain remains in concentrated urea. This gave mostly inactive material; after removing urea, a small amount of mercaptoethanol permitted rearrangement toward the native form. As an exact combinatorial check, eight labeled reactive sulfur groups have $7\times 5\times 3\times 1=105$ ways to be paired into four protein disulfide bonds: choose the partner of one group, then repeat with the unpaired groups. Only one pairing is the native one. Equal counting does not mean that all pairings are equally likely experimentally, nor that actual protein folding searches them uniformly. See Anfinsen's Nobel lecture, 1973 publication: scrambled ribonuclease and the 105 pairings, p. 223.

A Western blot detects selected proteins after their separation and transfer to a membrane, using antibodies. In the yeast cross-species complementation experiment, detection helps distinguish failed protein production from failure of an expressed protein to restore growth.

The useful idea is to preserve where proteins migrated in gel electrophoresis, then ask which positions retain an antibody. Western blot separation, Western blot transfer and Western blot antibody detection answer different questions. A band is evidence of retained label at a position; Western blot band interpretation requires Western blot controls before assigning identity or comparing abundance.

Western blot transfer moves separated proteins out of the gel onto a Western blot membrane, using an electric field across the gel thickness. Maintaining close contact preserves the lane pattern. Contact gaps, incomplete removal from the gel and passage through the receiving sheet can change how much protein is retained.

The original 1979 experiment pressed a wetted, $0.45\mu \mathrm{m}$-pore nitrocellulose Western blot membrane against the gel between scouring pads and plastic pipette-tray grids, held by elastic bands. The urea-gel assembly faced the cathode in acidic solution, with $6V/cm$ applied for one hour; the SDS-PAGE variant reversed polarity and used a glycine/methanol buffer solution. Stained gel/blot pairs checked pattern preservation. Corresponding labelled ribosome protein spots from two gels were compared through radioactive decay readouts after combustion. The stained gel remaining after transfer supplied a removal check; a second receiving sheet revealed breakthrough on overloading. These tests supported recovery for the examined urea-gel proteins, within experimental variation, while the SDS-PAGE results showed unequal staining recovery. Preserved band positions therefore did not establish universal quantitative transfer. ^[ref]