we can assert that synthesized polymeric ampholyte
contains in the structure except amino groups also phosphate groups.
The stock solution of acrylamide was prepared by mixing 30% acrylamide powder with 5.2% bis acrylamide, 10% NP-40, 10% CHAPS, 10% APS, 20mM NaOH as catholyte, 0.01[micro]M ortho phosphoric acid as anolyte, 8M urea and 250[micro]l of ampholyte
as sample overlay.
The isoelectric focusing of proteins can be determined using immobilized pH gradients or ampholytes
. In general, the pH gradient used for protein separation is in the range of pH 3-12.
After cooling to room temperature, samples were diluted with a solution containing 8 M urea, 4% CHAPS, 1% DTT, and 0.4% carrier ampholytes
and subjected to further sonication passages followed by 2-DE.
The protein solution was mixed with 0.45% (by volume) pH 2-4 ampholytes
, 0.45% (by volume) pH 9-11 ampholytes
, and 0.9% (by volume) pH 3-10 ampholytes
First-dimension isoelectric focusing was run on 4.1 g [L.sup.-1] acrylamide tube gel containing 9.5 M urea, 20 mL [L.sup.-1] Triton X-100, 5 mM (3[(3-cholamidopropyl) dimethylammonio]-1-propane-sulphonate), and 20 mL [L.sup.-1] Millipore 2D optimized carrier ampholytes
First, members of the Dpfp1 family have acidic isoelectric points ranging from 5.3 to 6.5; marine byssal precursors, in contrast, are highly basic - many with pIs exceeding the effective resolving range of available ampholytes
. Second, dreissenid byssal precursors, including Dpfp1, are glycosylated with N-acetylgalactosamine O-linked to serine and threonine residues; there is, however, no evidence for glycosylation in byssal proteins from any marine taxa.
Baker), 2% IGEPAL (Sigma-Aldrich), and 0.25% carrier ampholytes
In vitro-labeled samples were focused with wide range ampholytes
(pH = 3-10; Bio-Lyte 3/10, Bio-Rad) in 4 M urea and 10% NP-40, and size-fractionated on 10% sodium-dodecyl-sulfate poly-acrylamide gels (SDS-PAGE) along with Brome Mosaic Virus (BMV) molecular weight markers (110, 97, 35 and 20 Kd) provided as a control with in vitro translation kits (Promega).
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.
Many factors (e.g., variability in gel conductivity, quality of carrier ampholytes
, and electroendosmosis) make IEF inherently difficult to standardize.