Wittrup: FPLC general use

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These instructions were originally written by Katarina Midelfort in 1999. Please get trained by current Wittrup lab personnel before using for the first time. Some things may have changed.


The fast protein liquid chromatography (FPLC) instrument is from Pharmacia. It is comprised of pumps, switching valves, sample loop, separation column, UV detector, conductivity meter, sample collector, and plotter. The instrument is run from an internal computer.

Contents

Parts

The pumps: The pumps can run a constant percentage of two combined buffers or a gradient between the two buffers over time.

The internal computer: The instrument runs off an internal computer and can be run in manual mode or method mode.

The valves: The pumps are connected to valves which send the buffers in the desired direction: either directly to the waste, directly onto the column or directly through the sample loop and then onto the column.

The sample loops: The sample loops come in a variety of sizes based on your sample size. Use the sample loop closest to the volume of your sample. We have 25 ul, 50 ul, 100 ul, 200 ul, 500 uL and 8mL sample loops.

The column: The column is connected to the switching valve. Many different columns are sold for purification by different methods (ion exchange, hydrophobicity, size exclusion). We have a gel filtration column (Superose 12), a cation exchange column (Mono S) and an anion exchange (HiTrap Q). We also have some small testing columns in the drawer with the FPLC. Columns should be stored in 24% ethanol/H2O when not in use.

The UV detector: The UV detector is run from a small unit above the main FPLC computer and can detect 254 nm or 280 nm. It is usually set to detect 280 nm.

The conductivity meter: The conductivity meter is the small black tube connected after the UV detector. It is also adjusted from a small unit above the main FPLC computer.

The fraction collector: The fraction collector can be run or stopped by the main FPLC computer when running under a method file or manually on its own computer unit. It must be set for the size of the fraction to collect on its own computer either way.

The plotter: The plotter will print the results of the UV and conductivity meters as they get data and will make marks for when the fraction collector changes tubes.

General Use

To turn on the system, flip the red button near the top of the FPLC rack. This should turn on everything except the plotter and the UVis detector. The plotter is turned on by the switch on its side. The UVis has a similar flip switch on the back.

Put the red and blue pens in the plotter.

Push the manual button on the front panel and use the forward and backward buttons to see the variables that are possible to change.

To set a variable put in the new value in place of the blinking value and push enter. If you don’t wish to change the value just go forward or backward without changing the blinking value.

Standard settings:
conc. B 50.0 (therefore 50% of the fluid pushed through the system comes from pump A and 50% from B)
ml/min 0.5
cm/mL 1.0
PT 0.0.0.0.0.0.0.0.0
valve 1.1 directly to column
1.2 through loading loop and then to column- ONLY WITH SYRINGE IN
1.3 skips column and goes to waste- for wash
flow c 0.0
monitor0
level 0.0
ml/mark0.0
wash A.B1.0 to wash reservoir of A
0.1 to wash resevoir of B
1.1 to wash both reservoirs
0.0 no washing
integrate 0
feedtube
sync A.B 0.0
Chart recorder:
  • set to: ext
  • run at: mm/sec
  • chart speed: 20
  • set rec to: on
  • Have the pens “zero” down to set pens and up for the run
  • Put pens down for writing.
  • red-> conductivity
  • blue-> UV- 280nm
  • set for “cal”, not “var”
To wash pumps:

Use filtered degassed buffer only with the FPLC. Air bubbles on the column can ruin the column resolution.

Put the tube to pump A in bottle A and the tube to pump B in bottle B, or put both tubes in one bottle if you only have one buffer. Be sure they are always under the liquid level.

Set the valve to 1.3 and the flow rate to 5 ml/min.

Let this run until both pumps have fully equilibrated in the new solutions. Then stop the run.

To wash the column:

Connect a column to port 1 of the switching valves. Be sure that there is liquid in the tube directly connecting to the column so that no air enters the column. If the tube is dry then switch the pumps on to run to the column before connecting the column to get liquid in the tube. Let fluid run into any connecting nozzles to remove any air.

Connect the bottom of the column to the UV monitor tubing, again assuring not to force any air into the detector's flow cell. If you do end up with air in the flow cell, this may present as the detector signal jumping all over the place and the plotter flying up and down wildly. If this happens, use a syringe to force fluid through the flow cell to try and get the bubble out.

Set the ml/min to something low, like 0.5 ml/min and the chart recorder to 1 or 0.5 cm/ml

Watch the UV and conductivity meters and allow them to equilibrate, meaning that the conductivity monitor and detector signals stabilize.

Watch that the pressure does not go above 2 MPa with any size exclusion column, nor above 0.5 MPa with any ion exchange column.

You can increase the ml/min if the pressure remains low.

Trouble with columns:

If the back pressure on the column is high, there can be several problems. First, when ethanol is present in the column, the pressure will be higher than usual. This is fine, just use a low flow rate until the ethanol is flushed out. If the pressure remains high, the first place to check is the pre-column filter. Replace the filter with an extra from the FPLC drawer. If this does not help, then the flow cell my be clogged or the column may be dirty. Check column or UVis information sheets to determine cleaning solutions.

If the alarm is going off on the column for any reason, the machine will usually pause itself. Push the alarm reset button on the main computer to reset, then push continue (if you have figured out what caused the alarm and corrected it).

Loading a sample:

Connect a sample loop of similar size to the volume you wish to load on the column to ports 2 and 6 on the switching valve. Use a syringe with water in it to wash the sample loop by inserting the syringe into the syringe port on the switching valve. Push the syringe in all the way, but do not push it too hard. Inject the water through the sample loop, it will come out of a tube which should be held above the level of the loading syring so that the water does not drain out when done. Again, this is to keep air bubbles from forming in the sample loop which then might be injected onto a column.

Then load sample in syringe into sample loop. The sample volume should ideally be larger than the sample loop so that no air bubles will be present in the loop after loading. If the sample volume is smaller than the loop be careful that the water injected for cleaning is straight against the sample volume (no air bubble).

Leave the syringe in place after loading the sample loop. (leave syring in place during all times that the valve is switched to 1.2)

When the valve is switched to 1.2, the sample loop will be part of the flow to the column and the sample will inject onto the column. Once enough volume has gone through the loop to clear it out, switch the valve back to 1.1. Now you can safely remove the syringe and clean the loop (or reload the sample loop).

Running a method:

The gel filtration column is run with a constant buffer percentage and does not require a formal method to run. This can be run directly off the manual control and the fraction collector can be run manually as well (see info. about fraction collector below). However if you wish to write a method for it you may.

The ion exchange columns require a salt gradient for elution. This requires a method file. Method files are stored in 5 different method banks (1-5) and then in 10 different methods in each bank (0-9).

To set up a method:

There are some standard methods that can be loaded into the method bank 5 with the command Clear Memory 501. See inside the back cover of the controller manual for the standard methods.

I always edit one of the standard methods for my use when I need one. To edit a method, choose “method file” from the front panel. Then type 5 and enter for the 5th bank. Then use the forward button to get to “program method” and enter the method you wish to edit (example 0). Push “forward” to step through the commands of the method. An example with a few notes is below. The commands such as ml/mark and integrate have to do with the printing out of a small version of the run on the little onboard printer as the method runs.

Use the delete key to remove a command. Use the insert key to insert a command; once you have pushed the insert key you can scroll through the commands with the forward button until you reach the one you wish to insert, then hit enter. At this point you can enter the time you want the command for. Then use the change key to change the value of the newly inserted command. Use the change key to change the time or value of a specific command.

When you have the method the way you want it, choose exit from the front panel.

Now you can run your method by typing the method number and hitting enter (it will run the method of the method bank you were last in)

example of method:

0.00 conc %B 0.0
0.00 ml/min 1.00
5.00 valve.pos 1.2 this is to inject sample loop volume
5.00 port.set 6.1 this turns the fraction collector to run
5.00 clear data
5.00 monitor 1
5.00 level % 5.0
5.00 min/mark 2.0
5.00 integrate 1
10.0 conc %B 0.0
10.0 valve.pos 1.1 this stops going through th sample loop
30.00 conc %B 35.0
34.00 conc %B 100
38.00 conc %B 100
38.00 port.set 6.0 this stops the fraction collector
38.00 integrate 0
38.00 prt pk 1.05
38.00 conc %B 0.0
43.00 conc %B 0.0

To switch between the values being given in min or in ml, use the calibration key and in the first choice use 0 for minutes, 1 for ml, and 2 for dl. Then use the exit key to leave the calibration mode.

Trouble with a program:

As our Pharmacia system has aged, it has become prone to complete power and memory failures (frustrating I know). This will present as an error reading Check Code 190. This will require you to first reset the calibration on the pumps and the recorder by pressing the calibrate button on the controller and entering 110 for Pump AB and 200 for Rec. You will now be able to run manual methods and to reprogram old methods that have been cleared from the memory.

Fraction Collector:

The fraction collector can be told to run or stop from the onboard computer but can not be set for the size fractions to collect this way. The fraction collector is set by using its own key pad and I always use the simple time-based collection.

To set the fraction size:

  1. Press 1,9, then store return
  2. Press 7
  3. Press number for fraction size in fractions of a minute (based on your flow rate). example: 0.5 for half a minute
  4. Press store return
  5. Press run if you want to start it yourself (if not using a method) otherwise do not push this and leave it after step 4- the method file will start it when it reaches the port-set 6.1 command.

Put tubes in the fraction collector- use glass test tubes if collecting large fractions or put small eppendorf type tube in the glass test tubes if collecting small fractions (using the test tubes as holders for the small tubes). Set the exit stream tube over the first tube. The fraction collector will automatically move from tube to tube and will also mark the chart recorder for each tube moved.

When you are finished running the FPLC:

The column and system should be stored in filtered degassed 24% ethanol/H2O when not in use. Put this solution through the column at 0.5 ml/min until it is completely rinsed through. The pressure will rise when the ethanol is present in the column- this is normal. Disconnect the column from the FPLC and store capped (room temp is alright for many columns). Reconnect where the column was to keep the system from drying out.


Types of Columns (that we have):

Superdex 200 Size Exclusion

This column will separate proteins based on size. Larger proteins pass through fewer pores and thus pass through the column more quickly. This column if fairly flexible with respect to buffers. Run both pumps on the same buffer bottle and use a buffer with moderate buffering and salt concentrations depending on the characteristics of your protein. For this column and others based on exclusion chromatography use a sample size of 500 ul or smaller (the smaller the better). The exclusion limit for this column is 1.3x10^6 Da and the optimum separation range is 1x10^4-6x10^5.

Superdex 75 Size Exclusion

This column will separate proteins based on size. Larger proteins pass through fewer pores and thus pass through the column more quickly. This column if fairly flexible with respect to buffers. Run both pumps on the same buffer bottle and use a buffer with moderate buffering and salt concentrations depending on the characteristics of your protein. This column can be run in series with the other size exclusion column to achieve even better separation between proteins. The exclusion limit for this column is 1x10^5 Da and it displays optimal separation for proteins between 3,000 and 70,000 Da.

Anion exchange, HiTrap Q

This column will separate proteins based on how negatively charged they are. The more negatively charged the better they will bind. Use different pH buffers (usually 6-9) to find a pH that gives your protein enough charge to bind. Use a salt gradient, usually from 0 -1 M NaCl, to elute protein. Buffer A should be ~50 mM buffer at selected pH with no salt and buffer B should be the same ~50 mM buffer (same pH as A) with 1 M salt.


===== Replacement Filters

Bottom filter and O-ring for re-packing columns (GE Healthcare 19-5082-01) Top filter for columns, also for in-line filter (GE Healthcare Tricorn Filter Kit 10)

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