Editing Magnetic beads Preparation Methods (section)

== Cross-linked agarose&nbsp; ==

[http://www.jbc.org/content/254/8/2572.full.pdf http://www.jbc.org/content/254/8/2572.full.pdf]

A Novel Method of Activation of Cross-linked Agaroses with 1,l carbonyldiimidazole Which Gives a Matrix for Affinity Chromatography Devoid of Additional Charged Groups*&nbsp;

Cross-linked agarose (Sepharose CL-GB) was purchased from Pharmacia (Uppsala, Sweden), 1,1’-carbonyldiimidazole from Pierce (Rockford, Ill.), and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate from Merck (Rahway, N. J.). All other chemicals were of reagent grade and were used without further purification. Dioxane contained not more than 0.5% water. Subsequent to the completion of these studies, Pierce Chemical Co. has agreed to make the activated matrix commercially available as “CDI’-activated Agarose.” Trinitrobenzene sulfonic acid was purchased from Aldrich (Milwaukee, Wise.). Typical Activation Procedure-Sepharose CL-6B (3 g of moist cake) was washed sequentially with water, dioxane/water, 3:7; dioxane/water, 7:3; and dioxane (20 ml of each) and was suspended in dioxane (5 ml). CD1 (0.12 g) was added, and the suspension was shaken at room temperature for 15 min. It was washed with dioxane (100 ml) and used immediately. Coupling of Amines-The above material was treated at 4°C overnight with n-butylamine, 1,6-diaminohexane, or 6-aminohexanoic acid (1.4 g) in water (9 ml) at pH 10 and washed sequentially with water (200 ml), 1 M NaCl (100 ml), and water (200 ml). Materials for titration were further washed with 5 mu HCl (200 ml). Coupling of Ethanolamine to 6-Aminohexanoic Acid-SepharoseThe 6-aminohexanoic acid-Sepharose CL-6B (3 g of moist cake) was treated at room temperature with ethanolamine (0.5 g) and CMC (0.5 g), and the pH was adjusted to 6 and maintained overnight at that pH with a Radiometer pH titrator (TTTB). The final volume was 12 ml. The material was then washed as for the coupling step. Affinity Chromatography-Affinity chromatography was carried out on 0.4- to l-ml samples packed into Pasteur pipettes. The equilibrating, loading, and washing buffer was 0.05 M Tris (pH 8) and desorption was carried out with 3 mM HCl. All solutions contained 0.5 M NaCl. Loadings were 20 mg of trypsin in 16 ml of buffer for A, B, and D and 10 mg in 8 ml for C.