Updated FPLC Size Exclusion Procedure
From Heit Lab Wiki
- All buffers, 20% ethanol and samples must be pre-filtered using a 0.2 μm filter
- Install desired loading loop onto system before initiation, but do not install column
- Washing and equiliberating the system can take ~4 hours. If required, after equiliberization the system can be powered off overnight with the column attached. One column volume of buffer needs to be run through the following morning to ensure proper column equliberation.
Initiation & Purging System
- Turn on machine and allow to boot. Then start computer and open software.
- Check that the isopropyl lines are in a bottle of isopropyl alcohol, and that there are no airbubbles
- If air bubbles are present, unhook the outlet isopropyl line from behind pump #2 and attach the syringe adaptor. Using a syringe, pull isopropyl through the system until bubbles are gone. Reattach isopropyl line.
- Insert the input lines for both pumps into 20% ethanol
- Connect syringe to purge port on pump 1, and twist ~1/2 turn to open. Draw enough liquid through pump to purge lines of air bubbles. Close port and remove syringe. Repeat with pump 2.
- Set sample inject valve to "manual load loop", and inject 20% ethanol into the valve.
- 'NOTE: Insert blunt needle until resistance is felt - do not push further as this may damage the gasket.
- Inject until you see liquid dripping from the W1 waste line
- Double-click on pumps, set flow rate to 4 mL/min, 50% flow through pump B, isocratic mode, no pressure limit. Run until a consistent rate of dripping is seen from the waste line.
- Set sample inject valve to pump through loop, select fraction collector and turn on collection (place collector outlet into waste container to avoid filling tubes with waste), and run until a constant drip is observed through the fraction collector.
- Turn off fraction collection and change inject valve to system pump waste. Continue pumping at 4 mL/min until constant dripping is observed through waste line W2.
- Move pumps to distilled water and purge system as above.
- Set sample inject valve to manual load loop, and inject water until until drips emerge from waste line W1
- Set flow rate and pumps as per system flush, above, and run pump until constant rate of dripping is observed.
- Set sample injection valve to pump through loop, set fraction collector to collect fractions (again, direct output into waste to avoid wasting tubes) and run at 4mL/min until consistent dripping is observed through the fraction collector.
- Turn off fraction collector and set sample injection valve to system pump waste, 4 mL/min, until constant dripping occurs through waste line W2.
Wash Column With Water
- Attach column (Enrich SEC650) to system:
- Attach column to left side of machine using magnetic clips
- Remove "bridge" adaptor from column lines (brown lines emerging from sample inject valve) and plugs from column
- screw column plugs into bridge adaptor, and place somewhere where they will not get lost
- Connect column lines; line emerging from sample injection valve must go to top of column.
- Set flow rate to 1 mL/min, 50% pump B, max pressure of 600. Pump at least 1 column volume (30 mL [30 mins pumping] for SEC650 column).
- More time may be needed if column over-pressurizes and auto-reduces the flow-rate, which is not uncommon when clearing ethanol from the column. If over-pressurization occurs, try running at 0.75 mL/min.
- Move pump input lines into desired buffer (e.g. PBS) and repurge system.
- Manually equilibrate column with 5 volumn volumes of buffer, running buffer through the various waste lines and faction collector.
- Note: Machine can be powered off overnight at this step, and the run resumed the next day, if required
- Load 1.5 mL tubes with caps removed into fraction collector (this can be done during column equilberation
- Inject sample into inject valve. DO NOT REMOVE SYRINGE
- Run size exclusion program with no initial equiliberation step, check to make sure fraction size is appropriate (0.25 to 0.5 mL/fraction)
- Remove syringe once program indicates column loading is complete
- Samples will automatically be collected.
- Run can be stopped early once desired fractions are collected
- Remove fractions
- Save traces/CSV/etc at this point, as they may be lost during later steps
Requiliberiation & Washing
- Any residual protein must be purged from the column before cleaning. To do this, run 5 column volumes of the buffer used for sample fractionation through the column, using 1 mL/min, 600 max pressure, 50% pump B setting.
- Move pump lines to a container of filtered milli-Q water
- Purge pumps, set flow rate to 1 mL/min, 50% pump B, max pressure 600.
- Set inject valve to manual load loop, and inject milli-Q water until water comes out of waste line W1
- Set inject valve to pump through loop, select fraction collector and turn on collection (run output into waste), and run until a constand drip comes of out the fraction collector
- Turn off fraction collection and change sample inject to system pump waste. Run at 4mL/min until a continuous drip is observed through waste line W2.
- Set pump to 1mL/min and set inject valve to pump through loop, run at 1 mL/min through column until a column volume is run through column (30 mL).
Prep Column for Storage
- Place both pump input lines into 20% ethanol
- Purge pumps
- Run at 1 mL/min through column until 1 column volume has passed through column (30 mL)
- Unhook & cap column, place back into storage, and use bridge connector to link column lines together.
- Keep pump input lines in 20% ethanol
- Set loop to manual load, inject 20% ethanol until dripping is observed through waste line W1.
- Set pumps to 4 mL/min, 50% B, isocratic. Run until constant dripping is observed
- Set sample inject valve to pump through loop and set sample collector to collect samples (put output into waste), run a few mL through loop/sample collector.
- Turn off fraction collector and run a few mL through waste line W2
- Close software and shutdown laptop before shutting down machine
- Move machine monitor to a position where it is unlikely to be damaged