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Brendan Francis

Day 3: Size Exclusion Chromatography

Assignments Due

Preparation

Overview of Experiment

The salt pellet from Day 2 is run over the size exclusion column. This column serves as a desalting step by removing the ammonium sulfate and as a purification step by fractionating the proteins based on size. Activity of the deaminase is located using a QUALITATIVE spectrophotometric assay and active fractions are pooled for dialysis. Buffers for the ion exchange chromatography step are chosen, prepared, and used for dialysis.

View this animation on size exclusion (gel filtration) chromatography: Gel Filtration Chromatography: Fundamentals of Biochemistry by Voet, Voet & Pratt (Wiley)

Procedures

SEC Set-Up DEMO Videos (created by Xiao Zheng, Michael Yuan Yu Huang, and Xuan Yu, BIOC 311 Fall2011): Full-length (SEC Column Set-up) and 4 parts

Size Exclusion Chromatography

The column is part of a closed system when it is connected to the chromatography system, and a peristaltic pump controls the flow rate through the column (see Fig. 1). Two columns can be run from each peristaltic pump. One column at each station can be eluted through the UV monitor and an elution profile traced on the chart recorder (not shown in Fig. 1).


Fig. 1. Size exclusion chromotography station set-up.



Anion Exchange Buffer Preparation

As a team (3-4 students), design a buffer to use for ion exchange chromatography on the Bio-Rad Q cartridge. Determine the buffer composition and pH based on theoretical aspects of ion exchange and on the results of the stability study of adenosine deaminase.


Dialysis

The pooled sample from the size exclusion column (P-60 fraction) is dialyzed to change the buffer composition of the sample and decrease the volume for convenient application to the Q cartridge. Dialysis can take care of these two problems during the week without your attention. (Normally this is accomplished in several hours or overnight.)

  1. For dialyzing and concentrating your sample, prepare a solution of 10% (w/v) polyethylene glycol compound (recommended range = 15,000-20,000 MW) in 500 ml (TOTAL volume) of anion exchange buffer in a beaker. (The PEG does not have to be completely dissolved.) Because we are dialyzing over a weekend, the 10% solution is sufficient; for overnight concentrating, 20% PEG is recommended. The concentrating effect of this solution is sufficient to remove essentially all the free water and will leave only a precipitate of proteins in the collapsed tubing. Unlike some enzymes, the deaminase survives this treatment.

  2. Obtain a length of dialysis tubing, with a recommended range of 6,000-8,000 molecular weight cut off (MWCO), that will hold your sample plus 5 -10 cm excess.

    NOTE: Limit handling of the tubing with bare hands to prevent the contamination with enzymes and bacteria from your skin.
    The tubing box gives the molecular weight cut off limits and the volume of solution per cm of tubing (ml/cm).

  3. Soak the tubing in RO water for at least 3 minutes.

  4. Smooth out one end and place a clip over a single thickness of tubing.
    The clips are designed to close over only one layer of tubing; clamping more than this weakens the hinge and it may break during dialysis.

  5. With one clip in place fill the tubing with buffer or water to check for leaks over the surface of the tubing and the clip.

  6. After you have determined that the tubing is leak-free, discard the water and use a disposable transfer pipet to fill the tubing with your sample.

    Loading tip: hold the tubing over a clean weigh boat; if you spill your sample, you should be able to recover most of it.

  7. Apply the second clip, leaving some dead space in the tubing, and place the tubing into the dialysis solution set to gentle stirring over a magnetic stirrer in a cold box (4°C). Be certain that the stirrer will not pull the tubing into the bar as this may wear a hole in the sack.
    (A team may place both their samples in the same solution for dialysis.)

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 14 June 1999
Updated 20 June 2014