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Assignments Due

• Calculations in lab notebook (due at the beginning of lab)
  • Standard curve for Bradford assay and calculation of protein concentration for native and recombinant crude extracts
  • Volumetric flow rate (in ml/min) for size exclusion chromatography  NOTE: see step 5. under "setting up the column" below.
• Team library project
  • List of complete references and summary of library research findings (submitted as an "Assignment" in OWL-Space by the specified due date/time)
  • Elevator talk (2 minutes per team) *given during lab
• The St. Martin's Tutorial on Avoiding Plagiarism (10 points)
  
  • You must complete these ONLINE tutorials and exercises by 11:30 pm on Fri., 17-May-2013
• Introduction and References (McMillan: pp. 59-61, 78-81, 107-125, 126-160 (3rd ed.); 76-78, 104-107, 5-32, 149-153, 167-205 (4th ed.))
  
• Begin writing the Introduction section of the paper and compiling the References. Include relevant background information accumulated the first day of class to develop a reason for the study based on possible differences and a general outline of what approach your study uses to determine wherther or not the recombinant enzyme is the same as the native enzyme.
  
  Bring a TYPED draft of the Introduction with References (cited as in Protein Expression & Purification) to Day 4 lab; please use a paper clip; do not staple pages.
  See Research Paper .
• Protein Purification Reference
  1. Choose an article from any scientific journal that presents a purification of an enzyme AND contains a "purification table" (*see description below)
  2. "Photocopy" just the Materials and Methods section on purification and the purification section of the results (a purification table MUST be present in the article)
  3. Prepare a cover page with your name and lab section; record the complete reference for the article
  4. Bring this information with you to the 4th day of class
  
  *A purification table is a table that summarizes each step of the purification process and gives information such as the procedure used, total protein, total activity, specific activity, % yield, etc.
• Chromatography theory (Scopes 2nd ed. pp. 72-79; 3rd ed. pp. 102-111)
• Gel filtration (Scopes 2nd ed. pp. 186-198; 3rd ed. pp. 238-250)
• Ion exchange chromatography, including pH and buffer selection [Practical Aspects, Buffers for Use in Ion-exchange, Conditions for Absorption, Procedures for Elution of Proteins from Ion Exchangers] (Scopes 2nd ed. pp. 113-125; 3rd ed. pp. 157-171)
• Study Guide (bring a copy to lab)
• To familiarize yourself with operation of the Bio-Rad Econo System, look at the instruction sheet beneath each pump or visit Bio-Rad
• OWL-Space BIOC 311 Resources (bring a copy to lab)
  • [Buffer Charts]
    • ionXchange.pdf

Preparation

• 1st discussion
  • Size exclusion chromatography theory
  • Detection of ADA activity
  • Dialysis
• 2nd discussion (given while columns are running)
  • Elevator talks
  • Writing the Introduction

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 these animations on size exclusion (gel filtration) chromatography:
• Gel Filtration Chromatography: Fundamentals of Biochemistry by Voet, Voet & Pratt (Wiley)
• Gel Filtration Chromatography: GE Healthcare Life Sciences

Procedures

Assay stability samples

• Use the same assay methods as on Day 1 to determine the activity in the incubated samples.
• Compile your results to determine which buffers or conditions are favorable for use during purification of murine adenosine deaminase.
• As a team, design and prepare a buffer for dialysis and anion exchange chromatography.  Give a brief rationale for your buffer choice in your lab notebook.
  

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

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

• Preparing the sample
  1. Dissolve the ammonium sulfate pellet in a minimum of column buffer (normally the pellets can be dissolved to a final volume of 0.5 to 1.5 ml).
     SEC buffer:
     0.1 M KPO4,pH 7.4
     0.05 M NaCl
     0.02% Na azide
  2. Transfer the sample to a 1.5 ml tube and centrifuge (in the Eppendorfs) at maximum speed (16,000 x g) for 5 minutes to remove undissolved material (you may not see a pellet).
  3. Carefully transfer the supernatant to a clean 1.5 ml tube; store sample on ice until ready to apply to the top of the column.
  
  
• Setting up the column
  
  NOTE: Use ONE pump station per team (two columns run simultaneously)
  
  
  1. Install the columns in the rack with the spring loaded clips (see Fig. 2). Handle and position the columns carefully so as not to disturb the beads.
     *Remove any labels and return the clamps to the wooden cabinet and place the ring stand by the -80 freezer*
  2. Connect the outlet of the column to the fraction collector directly (column on the right) or through the UV monitor (column on the left).
  3. Open stopcock at the bottom of the column; put a small beaker under the arm of the fraction collector to collect the waste.
     
     
     

     Fig. 2. Positioning of SEC columns. As facing the columns, the one on the left connects to the UV monitor (the "black box").

     
     
     
  4. Carefully remove the buffer down to the level of the gel bed with a disposable transfer pipette (do not use mechanical pipettors). DO NOT DISTURB THE BEADS!
     NOTE: once the top of the beads appears "dry" close the stopcock until you load the sample.
     
     

     ***As soon as the samples are ready, LOAD them onto the columns (see "Running the column, steps 1-3" BELOW)--set the pump, fraction collector, and UV monitor WHILE the samples are draining into the beds. Remember, the rate-limiting step is getting the columns running!***

  5. Set the pump (see Fig. 3):
     • Calculate the volumetric flow rate for this column (diameter = 1.5 cm, length = 50 cm).
       Linear flow rates of 4-6 cm/hr are recommended for good resolution. Linear flow rate equals the volumetric flow rate (e.g., ml/hr) divided by the cross-sectional area of the column (with "r" in cm). Follow the instructions for the pump to set the flow rate.
     • Convert the units to ml/min; set this rate on the pump using the up/down arrows in the LED.
     • Make sure the tubing inner diameter is set to 0.8 mm.
     
     
     
     

     Fig. 3. Pump controls used for SEC. NOTE: do NOT press the toggle switches on the panels marked with a big, red X.

     
     
  6. Set the fraction collector (will be controlled "manually" today):
     • The fraction collector will rotate tubes under the drip spout at the time interval (in min) that you program on the front of the instrument.
     • Size exclusion columns typically will dilute the applied sample size by a factor of 5 to 10.
     • Ideally, you want your sample divided into at least 5 tubes for good resolution. TARGET 5 slices to capture the ADA peak (remember "area under the curve" from calculus)
       *Each team will decide the volume of each fraction for the native and cloned sources.
     • Put 40-50 plastic tubes in each fraction collector.
  7. Set the UV monitor (see Fig. 4) [only one column will be connected to the UV monitor today]:
     • Adjust AUFS on right side of monitor: set the RANGE to 0.2.
     • ZERO the monitor by pressing the left-most button; hold the button until it flashes.
     
     
     
     

     Fig. 4. UV monitor control panel.

     
     
  8. Put lines to the pump in a beaker of SEC buffer (200-250 ml is enough for both columns) and attach tubing from pump to caps.
     Hold caps over beaker and press the purge ("fast" running man) button to fill the lines with buffer; press button again once lines are filled to turn off the purge mode; drape tubing with caps over edge of beaker.
     

     Do NOT use purge when the lines are attached to the column.

• Running the column
  1. To load the sample:
     • make sure the stopcock is OPEN
     • with a disposable transfer pipette, slowly draw up the sample from the 1.5 ml tube
     • layer the sample carefully onto the upper bed surface, keeping the tip against the column walls and moving in a circular motion (minimize generation of "bubbles" as these can denature proteins) NOTE: never disturb the top of the bed by dripping or squirting liquid into it
  2. AFTER the sample drains completely into the bed, add buffer (0.5 to 1 ml) to wash the sample into the bed (this step is called the "chaser").
  3. AFTER the chaser has entered the column, layer fresh buffer to a height of 3-5 cm above the bed and attach the cap (do not put cap on tightly).
  4. START the PUMP ("slow" running man) at the flow rate calculated above.
     
     

     Once you've started the pump, DO NOT STOP it until the team has collected ALL the fractions with ADA activity for both native and cloned.

  5. Swing the arm of the fraction collector over the tubes and start the fraction collector (push the "running man").
     
     NOTE: when you start the fraction collectors, please ask us to start the chart recorder (paper speed = 6 cm/h) for you.
     
     Suggestion: While the column is running, familiarize yourself with the chromatography station. Read through the manual for the controls for gradient formation, fraction collector, column injection, etc. You will be running a cartridge column using a gradient next lab period that will employ nearly all the features of the station.
     
     ***Record the bead height of each column to the nearest cm. Wait until the columns have been running for a while since the bead height will decrease because of packing.***
     
     
  6. Test for adenosine deaminase activity in the fractions using the qualitative spectrophotometric assay described below. Combine all the fractions with measurable activity. NOTE: Save a 200 µl aliquot, label, and store at -20 degrees C.

     ***This spectrophotometric assay for ADA activity is similar to the one used for the Stability Study. Since only QUALITATIVE information is required for this lab, the assays can be run "sloppily." Always use the ADA assay buffer (50 mM KH2PO4, pH 7.4) for both quantitative and qualtitative ADA assays.***
     
     You MUST use a Genesys 5 Spectrophotometer for the QUALITATIVE ADA assay.

     1. Use 1. Absorbance from the main menu and set the wavelength to 265nm using the "GO TO WL" key; type in 265 nm then press "ENTER."
     2. AUTO ZERO the spec with 3 ml of KPO4 assay buffer (place the cuvette in slot 1).
        [Why do you AUTO ZERO the spec BEFORE adding the adenosine?]
     3. Add 30 µl of 10 mM adenosine and invert to mix.
     4. Add 20 µl of the column fraction, invert to mix, and place the cuvette in slot 2. ADA activity is indicated by a steady decrease in absorbance. At least ten fractions with NO activity can be screened in a single cuvette. Begin at one end of the rack and proceed until activity is found. Make a new solution in a cuvette and start assaying fractions from the other end of the rack to bracket the activity. One or two fractions within this bracket should be tested to ascertain that it is a true and continuous peak of deaminase activity.
     
     
  7. Take a reading of the combined fraction at 280 nm on the spectrophotometer. Use the size exclusion column buffer to ZERO the instrument. This is a case when it is advisable to use the same cuvette for zero and the measurement because small variations between the cuvettes add error to the measurement.
     
     
     • Genesys 5 spec: Select "1. ABS/%T/CONC" from the main menu and use the "GO TO WL" key to set the wavelength to 280 nm. Place the column buffer in slot 1; close the lid and press "AUTO ZERO" -- confirm that the solution reads zero by placing the cuvette in slot 2 (DO NOT PRESS "MEASURE"). Replace the buffer solution with your protein solution, put the cuvette in slot 2, and read the absorbance on the screen (DO NOT PRESS "MEASURE").
       
       
     • Biowave spec: Press the "Other" key followed by the "Single/Multi λ" key. Select "Single λ", then click on "Set λ" and set the wavelength to 280 nm. Place cuvette containing column buffer to the far-LEFT side of the chamber; press "REF." Replace the buffer solution with your protein solution, put the cuvette to the far-LEFT, and press "TEST."
       
       
     • Libra S22 spec: Press "1" on keypad to enter Basic Modes. Press "1" on keypad to select Absorbance; set the wavelength to 280 nm and press F3 ("OK"). Place cuvette containing column buffer in the BLUE cell (cell 1), close the lid, and press green run key. Replace the buffer solution with your protein solution, put the cuvette in the BLUE cell (cell 1), close the lid, and press green run key.

     SAVE YOUR PROTEIN SOLUTION!!

     Optimally, one determines protein concentrations and activities associated with the individual fractions to aid in the decision of what fractions are to be pooled and which are to be discarded. The absorbance trace from the elution profile can be used to estimate the relative amount of protein in each sample. A280 readings are quick, nondestructive, and provide a good estimate of protein concentration in fractions.

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 over the weekend 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, 25 May 2010
Updated 3 January 2014