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Day 5: Anion Exchange Chromatography

Assignments Due

Preparation

Overview of Experiment

Run ion exchange columns using a salt gradient to elute adenosine deaminase. Complete all the measurements needed to complete a purification table for your enzyme preparation including activity measurements for all stored samples and the Q-cartridge fractions. Determine protein concentrations for all samples.

Procedures

The chromatography systems have been plumbed for running the ion exchange cartridge. All components of the station will be used except for the "peak collection" option, marked with the red "X" (see Fig. 1). Each team will share an instrument. The same conditions should be used for both native and cloned preparations and the samples will be run sequentially. Construct a 0.5 M salt buffer (250 ml is sufficient volume): add salt to your anion exchange buffer.


Fig. 1. A) Pump settings for anion exchange chromatography.

B) Q cartridge hook-up.

Preparation of Sample

  1. Wearing gloves, rinse the outside of the dialysis tubing with RO water.
  2. Recover the residue from the dialysis tubing with a MINIMUM of anion exchange buffer.
    A total final volume of less than 1 ml is recommended. Achieve a more quantitative recovery by rinsing the tubing with more than one addition of fresh buffer (e.g., three rinses with 0.3 ml or two rinses with 0.5 ml are better than one addition of 1 ml).
  3. Mix the recovered suspension well to dissolve the residue.
  4. Centrifuge in a 1.5 ml tube for 5 minutes at maximum speed to pellet any insoluble material.
  5. Transfer the supernatant to a clean 1.5 ml tube.
    The sample is ready for loading onto the Q cartridge.

    Caution: Do not shake to cause foaming while attempting to dissolve the material. Foaming indicates the presence of denatured protein that will act as a detergent to denature even more protein.

Q Cartridge Set-Up

NOTE: Use ONE pump station per team and run columns sequentially.  Use ONLY the "slow-running man" when operating the pump today (i.e., do NOT ever use the "fast-running man" = purge).

***Set the UV monitor: select 0.05 for RANGE; ZERO the monitor as on day 3.***


  1. Install your buffers in the chromatography system (Fig. 2). Buffer "A" is anion exchange buffer (the loading buffer) and Buffer "B" is anion exchange buffer with 0.5 M salt (the eluting buffer). NOTE: the pump tubing diameter is 1.6 mm.

    By DEFAULT, the pump pulls through the Buffer A line; to switch between buffers A and B, press the "Gradient Former" button while the pump is running.


    Fig. 2. Buffer set-up.


  2. Set the flow rate to 6 ml/min.
  3. Disconnect the top of the Bio-Rad Econo-Pac Q cartridge (5 ml) from the system and flush the lines with both buffers (this step will only take a few seconds).
  4. Reconnect the Q cartridge and rinse with Buffer B for 3 minutes then rinse with Buffer A for 3 minutes (flow rate = 6 ml/min). The column is now equilibrated in your starting buffer. You will need to rinse the cartridge again between sample loadings.

    Remember to return to the desired flow rate before loading the second sample.

  5. Set the flow rate for your run; Bio-Rad recommends 0.5-3 ml/min for the 5 ml Q cartridges.
  6. Program a gradient of no more than 30 minutes; you can program up to FIVE steps (Fig. 3).

    Fig. 3. Setting the gradient: A) Selection of "time" for a given step.

    B) Selection of "% b" (Buffer B) for a given step.

  7. Set the fraction volume ("test tube") using the pump (*you do not need to set Vo, Vt, or # fractions), zero the UV monitor, and activate the pump (turn "on" using the slow running man).
    NOTE: Make sure you see a "dashed, red line" on the fraction collector LED (see Fig. 4).


    Fig. 4. Appearance of the fraction collector when it's controlled by the pump.



  8. Be certain the injection valve is in the "Fill" position (CCW) then load your sample into the injection loop (maximum of 1.3 ml) using the syringe (see Fig. 5A).
    DO NOT REMOVE THE SYRINGE FROM THE PORT!!
  9. Check that the fraction collector is ready and the chart recorder is on; then move the injection valve to the "Inject" position (CW), and push the blinking "Inject" button to start the gradient (see Fig. 5B).

    Fig. 5. Sample loading: A) FILL the LOOP (CCW position).

    B) INJECT the COLUMN (CW position).

    Observe that the sample is being rinsed into the cartridge and that the fraction collector is started. Monitor the progress through the first two or three tubes to be certain of function and alignment of the fraction collector.

  10. Use the Qualitative Spectrophometric Assay for ADA Activity (SAME as on Day 3) to find the ADA activity in your fractions.
    *YOU MUST USE THE SPECTRONIC GENESYS 5 SPEC'S FOR THIS ASSAY.
    **ASSAY ALL FRACTIONS!

  11. Combine the active fractions and obtain an A280 reading for the combined fraction as you did on Day 3; this value will be used to determine protein concentration. What buffer do you use to AUTO ZERO the spec?

    SAVE YOUR PROTEIN SOLUTION!!

  12. Use the ADA activity spectrophotometric assay at 265 nm, as used on Day 1, to obtain QUANTITATIVE assessment of the activity in EACH STEP of the purification (NOTE: you do NOT need to use a CONTROL enzyme). Thaw the aliquots from past steps and keep them on ice. Some samples may have too much activity to measure even using only 1 µl. Be prepared to dilute a small fraction of the aliquot to add at least 10 µl to the assay for the activity measurement; do NOT dilute ALL of the fraction aliquots. On the other hand, some samples may have very little activity in just 20 µl; you may add up to 100 µl of fraction in the assay.

    Obtain activity measurements for crude fraction, P-60 fraction, and Q fraction for BOTH native and cloned sources:
  13. Add necessary components for stability (e.g., glycerol, DTT, beta-mercaptoethanol, sucrose, EDTA) to the final purified enzyme fraction.
    NOTE: a 0.5 M EDTA stock solution has been prepared for your use; you do not need to weigh out DTT--just use a "speck" on the end of a small spatula.
  14. Store final purified enzyme fraction in 5 ml tubes with screw caps at either 4°C or -20°C.
    NOTE: set aside 50µl of your Q fraction for desalting (see procedure below).

Desalting of Q Fraction for SDS-PAGE

All samples need to be in similar buffer prior to loading the SDS-PAGE gel (Lab Day 5).
The buffer composition of the Q fraction must be changed; the sample will be desalted using small columns equilibrated in the Tris/glycine/SDS running buffer.

    Quick method for changing buffers of samples for SDS gels:
  1. Obtain an Ultrafree (0.22 µm) device for each sample to be processed and insert the column into a 1.5 ml centrifuge tube.
  2. Using a disposable transfer pipet, fill the upper portion of the Ultrafree device with a slurry of G-25 beads (about 400 µl).
  3. Pulse spin (hold the "SHORT" key on the microcentrifuge) for ~15 seconds in the microfuge to remove excess water; discard the flow-through (waste water).
  4. Add 100-200 µl of Tris/glycine/SDS running buffer to the gel material and let stand for 1-2 minutes.
  5. Remove the excess buffer with a 15-20 second pulse spin.
  6. Carefully pipet 50 µl of your Q fraction on the gel material and let stand 2-5 minutes at room temperature.
  7. Recover the sample in a clean 1.5 ml microcentrifuge tube with a 10 second pulse spin.
    The sample will be in an acceptably desalted state.
  8. Store the sample at -20°C (*do NOT put "loose" tubes in the freezer--use a screw-cap bottle).
  9. Rinse out the beads with RO water and RETURN the Ultrafree device to the container.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 May 2010
Updated 5 May 2012