Editing Magnetic beads Preparation Methods (section)

== Agarose Preparation Protocol 2 ==

'''Preparation of Uniform-Sized Agarose Beads'''

A uniform sized agarose gel was prepared by emulsification technique, where 4% agarose was used as water phase and liquid paraffin was used as oil phase. Additionally, 2.5 wt% Tween 80 was added as surfactants which might help to prevent the coalescence of agarose gel. So agarose was dissolved in distilled water with containing 0.9% NaCl and the solution was heated by microoven to a temperature above the gelation point of the agarose. The solution was designated as water phase. The mixture of liquid paraffin and Tween 80 was heated to a temperature above the gelation point of the agarose approximately 80°C and the solution was designated as oil phase. The oil phase was charged into the water phase and agitated quickly by agitator. A uniform sized emulsion was formed in the reactor. The emulsion was added into second hydrophobic liquid cooled paraffin at -20°C to solidify the agarose droplets into beads. Finally the agarose beads were recovered from the second liquid and washed with distilled water. After washing the gel was screened, standardized mesh No. series as 25, 60, 120, and 270 was used to determine the particle size distribution of a granular material. After screening, the obtained uniform size gel was sent for microscopic view.

'''Cross-Linking of Agarose Gel by Epichlorohydrin'''

About 50 ml uniform-sized agarose gels were taken in a 250 ml beaker containing 20 ml of 2 M NaOH and 5ml of Epichlorohydrin. The mixture was allowed to react for 2 hours at 40°C with constant mixing in a home made rotary mixture to keep the concentration uniform [17]. After reaction, the gels were washed with deionized distilled water until it became neutral (conforming by litmus paper). 2.3. Modification of Agarose Gel with 1,6-Hexanediamine For the modification of agarose gel, 300 ml 0.2 M of 1, 6 hexadiamine solution containing 1.5 gm sodium carbonate decahydrate was mixed with the epoxy cross-linked agarose gels. The reaction was carried out at 60°C for 2 hours [18]. After reaction the gels were again washed with distilled water until the gel became neutral.

'''Activation of Agarose Gel with 2,4,6-trichloro-1,3,5-trizine'''

Amino modified agarose gel was soaked in 50 ml of 3 M NaOH solution. It was placed in ice bath and 150 ml of a 0.5 M 2,4,6-trichloro-1,3,5-trizine solution was mixed with homemade mixture in refrigerator for 60 mins. After activation, the gels were rinsed successively with 500 ml of 50 vol% acetone aqueous solution and 2 liter distilled water was used for washing to remove the unreacted 2,4,6- trichloro-1,3,5-trizine.



'''Immobilization of ConA to the 2,4,6-trichloro-1,3,5-trizine'''

Activated Gel 2,4,6-trichloro-1, 3, 5-trizine activated gel(collected from title 2.4.) was placed into an ice bath and mixed with 0.1 M sodium phosphate buffer, pH 7.8 containing 200 mg of commercially conA with continuos mixing for 12 hrs. After coupling, the gel was separated and 150 ml of a 0.5 M ethanolamine aqueous solution was mixed for 8 hrs to block the unreacted triazine groups. Finally the gels were rinsed with 1 liter distilled water [19]