, Pharmalyte pH 6-8 and pH 7-9, were obtained from Pharmacia.
9% (by volume) pH 3-10 ampholytes
Each solution was then dried by centrifugation under reduced pressure, after which 180 =L of rehydration buffer (7 mol/L urea, 2 mol/L thiourea, 20 g/L CHAPS, 50 mmol dithiothreitol, and 5 mL/L ampholytes
, pH 3-10) was added to the dry sample to solubilize and denature the proteins.
Each serum sample (500 [micro]g of protein) was solubilized in 8 mol/L urea, 40 mL/L CHAPS, 40 mmol/L Tris, 2 mL/L Bio-Lyte 3/10 ampholytes
(Sequential Extraction Buffer 2; Bio-Rad) with 5 mmol/L tris(2-carboxyethyl)phosphine reducing agent and incubated for 1 h at room temperature.
variability in gel conductivity, quality of carrier ampholytes
, and electroendosmosis) make IEF inherently difficult to standardize.
In addition, sequestering of iron by the ampholytes
during IEF might also occur.
, pI range 3-7, at constant power (15 W) for 1 h at 4 [degrees]C (refrigerated circulating bath).
In reality, the pH gradient created by ampholytes
is subject to disturbances by sample constituents that lead to deflections of the lines of equal pH (iso-pH) and produce bands that have been characterized as "frowns" or "smiles".
Urea, Ready-Mix IEF acrylamide/bisacrylamide (29:1 by weight), ammonium persulfate, and N,N,N,N-tetramethylethylenediamine were purchased from Amersham Biosciences, and the ampholytes
Servalyt 2-4, 4-6, and 6-8 were purchased from Serva.
Serum containing ~5 [micro]g of total AFP was applied to a mixture of 50 g/L sorbitol, 100 mL/L glycerol, and ampholytes
For example, isoelectric focusing (IEF) in immobilized pH gradients is compared with IEF in carrier ampholytes