Five IPG Immobiline DryStrips 24 cm, pH 3 to 10 (GE Healthcare, Bucks, United Kingdom) were rehydrated overnight in 450 [micro]l 2DE rehydration buffer (7M urea, 2M thiourea, 2% CHAPS, 0.5% pH 3 to 11 ampholytes
(GE Healthcare), 1% DTT, trace bromophenol blue).
Protein samples (1000 mg of total soluble protein) were diluted in 350 mL Destreak rehydration solution (GE Healthcare), supplemented with 0.2% ampholytes
3-10, and applied to IPG strips (pH 3-10 NL -17 cm, Bio-Rad, USA) by in-gel rehydration.
For each sample, 350 [micro]g of venom proteins homogenized in rehydration buffer (7 molx[L.sup.-1] urea, 2 molx[L.sup.-1] thiourea, 1% CHAPS, and traces of bromophenol blue) have been used with DTT 40 mmolx[L.sup.-1] and 0.5% ampholytes
at pH 3-10NL with a final volume of 250 [micro]L.
For two-dimensional electrophoresis (2D) the IPG strips (17 cm and pH 4-7) were passively rehydrated during 16 h with 300 [micro]L hydration buffer (8M urea, 2% CHAPS, 50mM DTT, 0,2% ampholytes
3-10, and 0,001% bromophenol blue) containing 10 [micro]g of the high abundance proteins or 15 [micro]g of the low abundance proteins.
Immobiline DryStrips (length, 18 cm; GE Healthcare, UK), containing immobilized ampholytes
forming a linear pH gradient of 4 to 7, were used for isoelectric focusing (IEF) in the first dimension.
The separation of different haemoglobin (F, A, S, and other types of haemoglobin) was obtained after application on thin layer home-made agarose gel containing ampholytes
pH 6-8 (ref.
Samples were further adjusted for IEF fractionation by combining 900 [micro]L pooled sample with ampholytes
(150 [micro]L, pH 3-10; Novex, Thermo Fisher, ZM0021; Waltham, MA), dithiolthreitol (DTT; 25 [micro]L, 4 M), and bromphenol blue (20 [micro]L, 10mg/mL).
The isoelectric pH gradient (IPG) tube gel was prepared using 8 M urea; 4% acrylamide; 2.4% ampholytes
; 2.4% CHAPS; and NP40 according to Protean IEF Cell (Bio-Rad, USA) manufacturer's instructions.
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Extraction of proteins: Approximately 1 g of pulverised muscle tissue was homogenised (Wiggen Hauser D500, USA) in 3 mL of ice cold extraction buffer containing 7M Urea, 2 M Thiourea, 50 mM DTT (dithiothreitol), 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS), 0.4% (v/v) carrier ampholytes
(pH 3-10, BioRad, USA) and 50 L of protease inhibitor cocktail (Catalogue No.
Analytical method: For sample processing, was used the technique of the isoelectric focusing electrophoresis (IEF), in which the separation of hemoglobin was performed by loading a blood sample on an agarose gel containing ampholytes
with a pH between 6 and 8 and quantified by densitometry.
The isoelectric focusing (IEF) was performed without ampholytes
and glycerol according to the manufacturer's instructions for peptide focusing.