Embryos of 25 40 mg (fresh weight) were excised under sterile conditions.
Ten immunoreactive spots with low molecular weight were excised manually from the Coomassie blue-stained gels.
Bands of the expected molecular weight were excised, purified and ligated into a plasmid vector using Topo-TA kits (Invitrogen).
PCR products of the correct molecular weight were excised after electrophoresis on an agarose gel and purified using Qiagen Gel Extraction kits (Qiagen, Inc .. Big Dye (Applied Biosystems Inc).
Bands with the expected molecular weight were excised from the agarose gels, purified with SpinX column (Costar) and cloned in pGEM-T vector according to the manufacturer's recommendations (Promega, Madison, WI, USA).
Body weights were measured, tumours were excised, tumour weight and diameters were subsequently determined and the presence or absence of peritoneal carcinomatosis was noted.
Subsequently, 18 equal slices, covering the largest polypeptides (slice number 1, molecular weight > 94,000) to the smallest polypeptides (slice number 18, molecular weight < 14,000) were excised from the stained gel for both samples.
Briefly, stained bands corresponding to protein of molecular weight 58 kDa were excised, cut into small (<1 mm3) pieces and washed three times by repetitive dehydration and hydration using, respectively, 100% acetonitrile (MeCN) and 100 mM ammonium bicarbonate (Ambic).
Mice were euthanized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg body weight), and hearts were excised and weighed.
