| | Re: Removing Albumin from Samples
Electrophoresis method to albumin remove from mixtureA PAGE, in the absence of denaturing reagent, was performed using Bio-Rad Slab Gel Electrophoresis system according to the manufacturer's instructions. Slab gels consist of a 7.5 % acrylamide resolving gel and a two parts of 4.5 % stacking gel respectively : a) load gel and b) Blue sepharose CL-6B gel. On the total length of the gel 14.8 % is load gel, 11.11 % blue sepharose CL-6B gel, and 74.1 % belongs resolving gel. The stacking gel containing Blue Sepharose CL-6B (Pharmacia) is constructed on top of the resolving gel and is prepared dissolving, with gently mix, 0.09 g of Blue Sepharose CL-6B per ml of stacking gel. Without delay, the slab gel plate is filled with the gel mixture. To avoid sedimentation of the Sepharose, the volume of ammonium persulfate is adjusted to obtain gel polymerization within 2 min. After polymerization, the stacking gel mixture without Blue Sepharose CL-6B is poured on top and a comb is inserted immediately to construct sample wells. Samples are prepared mixing 5-10 Ál of serum o supernatant of culture with 40 Ál of Tris-HCl 10 mM at pH 6.8 containing 20 % glycerol and 0.002 % of bromphenol blue. Twenty microliters of this sample is loaded into a well and initially electrophoresed at a current of 20 mA. After the bromphenol blue dye front had moved about 1 cm into the resolving gel, the current is increased to 40 mA. When the dye front reached the bottom of the gel, electrophoresis is stopped, stain with Coomassie brilliant blue R250 for protein detection, or transfer blot proteins by means electroblotted (Bio-Rad) onto a nitrocellulose membrane (Bio-Rad) for Western blot assay without albumin interference.