WEB-BASED SKILL 7

PROTEIN PURIFICATION USING COMPUTER SIMULATION.

1. Column chromatography

As we have seen, the isolation and characterisation of proteins is one of the most important
techniques in biology, and this process usually commences by using a relatively crude
fractionation techniques based on breaking the hydrogen bonds between the solvent (usually
water) and the protein. Thereafter a series of more sophisticated purification techniques based
on chromatography facilitate the isolation of INDIVIDUAL proteins. Last time we explored
the basic effects of temperature and salting out on protein fractionation. At any stage in the
purification process the continued presence of an enzyme canbe monitored by using an
enzyme assay. It can also be identified using 1-dimensional and/or 2-dimensional
polyacrylamide gel electrophoresis. Moreover, if a specific antibody is available the protein
can be identified in a gel using a specific antibody to visualise it.

Today we are going to make a much more specific approach to purifying our enzyme of
interest using the special software designed and made freely available by Andy Booth of
Leed’s University. This software can be found here http://www.agbooth.com/pp_ajax/ I
suggest that you use it in the on-line mode.

Also, please access the following web sites, and watch the videos
Ion exchange chromatography: http://www.youtube.com/watch?v=q3fMqgT1do8
Gel filtration Chromatography: http://www.youtube.com/watch?v=oV5VB5kO3tQ
Affinity Chromatography: http://www.youtube.com/watch?v=FUAQKjKT99Y
Visualisation of the physical experiment: http://www.youtube.com/watch?v=zAgr-6Q9mQ4

The separation software can be found here http://www.agbooth.com/pp_ajax/ Use it in the

on-line mode. Please begin this exercise by accessing the ‘Help’ menu and carefully working
through the first 5 examples.

Use this box to note down key aspects of the parameters that are exploited in order to purify
proteins and to check the results of such processes.

Now use the information from these web-links to provide explanations as to the usefulness of each
technique in the table on the next page.
 TERM USEFULNESS IN PURIFYING PROTEINS.

Ammonium Sulphate.

Temperature.

Gel filtration
Chromatography

Ion exchange
chromatography

Affinity Chromatography

Isoeletric Point

2-D Gel Eletrophoresis.

Antibody.

Spectrophotometer.

PREPARATION REFLECTION
(See PRACTICAL GUIDELINES 1.4 for advice on how to complete this section).

1. Now access the QMplus home page for this course, click on ‘Practical and Lab-based
skills’ and open the file entitled: Protein purification and find your name. Note the
number of the protein you have been asked to purify.
1. Return to the protein software and drop down the ‘Start’ menu at the top left-hand
side of the window and select ‘Start from the beginning’, and then ‘Complex Mixture’
followed by’OK’.

2. Select your designate protein followed by ‘OK’. The description of the protein should
be noted because it provides a possible first crude separation step in the purification
process. Use this information to decide on which process(es )you wish to subject the
mixture to in order to differentially precipitates the proteins (pH, temperature, salting
out).

3. Drop down the ‘Separation’ menu and select the first step purification step that you
wish to use. Explain the precise set of conditions you used and why.

4. Now check the results using an appropriate PAGE system.

5. Print* this off and insert it into your book. Comment on your findings.

6. Use the information from the PAGE result to decide upon your next purification step.
Explain your choice of system.

7. Now drop down the PAGE menu and select ‘Hide Gel’.

8. Use the ‘Separation’ drop-down menu to select the net process.

9. If you eventually opt for chromatography of soe sort identify the fractions containing
your enzyme using the assay on the ‘Fractions’ drop-down menu.

10. Print* this off and insert it into your book. Comment on your findings.

11. Use the ‘Fractions’ drop-down menu and select ‘Pool’. Use the selector facility to
pool the fractions of interest. The yield is then recorded in the automatic running total
list.

12. Repeat 5 to 10 until you have a gel with just ONE protein spot.

13. Print* this off and insert it into your book. Comment on your findings.

14. Comment on your purification process in terms of recovery, purity and cost. Might
you be able to do this more cheaply/efficiently?

* Please see over the page for a detailed explanation of how to record your results.
HOW TO RECORD YOUR RESULTS
1. Use the ‘Print Screen’ facility to capture the entire web page:

2. Use the ‘crop’ to edit the ‘Printed Screen’ and then the drag facility to enlarge it to provide
a figure like:
 INSERT EDITED SCREEN PRINT FOR STEP 5 HERE

PROVIDE COMENTS HERE.

INSERT EDITED SCREEN PRINT FOR STEP 10 HERE

PROVIDE COMENTS HERE.

INSERT EDITED SCREEN PRINT FROM STEP 13 HERE

PROVIDE COMENTS HERE.

DISCUSSION (Use the information on the summary sheet of your purification process )
COMPLETION REFLECTION.
(See PRACTICAL GUIDELINES 3.4e in the practical booklet for advice on how to complete this
section).

This is NOT an acceptable attempt because….

PLEASE SAVE AS A PDF FILE AND UPLOAD TO QMPLUS.