AKTA Prime: Cleaning pumps prior to run

(with or without column connected)

Place both tubes into 100% Reagent Alcohol.

From the <main menu> OK → <Templates> OK → <Application Template> OK→ scroll 12 steps to < System Wash Method> OK → < Select Buffer V. Pos> move

B; A: -,-,-,-,-,-,-,

curser all the way to right and press OK (this chooses both tubes to uptake alcohol → < start run > OK → hear beeping - an error always occurs press ESC and then Pause/Cont. to continue run.

78 ml of ethanol will be moved thru FPLC. 75 ml goes directly to waste, 1 ml thru column then to waste, 2 ml goes thru column and the exits out fraction collector.

Redo exactly the same protocol with water. Must be filtered and degassed if column connected.

AKTA Prime: Column Cleaning Protocol

Put both tubes in either degassed 1 M NaOH or degassed and filtered 2 M NaCl. (NaOH cannot be filtered.)

Purge air from the system before connecting the tubes to the column. Do this by starting method and allowing tubing to drip before connecting it to the column.

< Main Menu> OK → < Templates > OK → < Method Template > OK → < Gel Filtration/ Buffer Exchange > OK → < Inject …> OK (not necessary to change) → < Set Pressure > OK → change to 0.3 (for any column) OK → < Set Flow Rate > OK → change to 2.0 ml/min OK → < Set Fraction Size> OK → change to 400.0ml OK (volume should be larger than column volume → < Set Equil. Volume > OK → change to 1.0 ml OK→

[ Note: you could alternatively change the fraction size to 1.0ml and the equilibration volume to 400.0 ml. This will make liquid run to waste whereas now it all exits via the fraction collector in one large fraction.]

< Set Sample injection volume> set to lowest since no sample will be injected → <elution volume> set to 400 ml.

This method was saved as method 2 with the equilibration volume set to 400.0 ml and the fraction size set to 1.0 ml.

→ < Start Run> OK → hear beeping from error press ESC and then Pause/Cont. to continue run.

Repeat run with filtered and degassed water

Repeat run with filtered and degassed 20% ethanol (using the 200 proof ethanol) for column storage.

AKTA Prime: Alternative Column Cleaning Protocol

Put both tubes in either degassed 1 M NaOH or degassed and filtered 2 M NaCl. (NaOH cannot be filtered.)

Purge air from the system before connecting the tubes to the column. Do this by starting method and allowing tubing to drip before connecting it to the column.

< Main Menu> OK → < Manual Run > OK → < Set Flow Rate > OK → Scroll up to 2.0 ml/min OK → < Set Pressure > OK→ scroll up to 0.3 OK → < Start Run > OK → Beeping error- press ESC then Pause/Cont.

This program will dump into waste until someone stops the program. It will NOT stop automatically and must be stopped by someone before the liquid runs out.

Repeat run with filtered and degassed water

Repeat run with filtered and degassed 20% ethanol (using the 200 proof ethanol) for column storage.

Be sure that the volumes run thru column are larger than the column volume to fully wash column and exchange into 20% ethanol.