Exercise 9.

Affinity Chromatography
Pure proteins are required for a wide variety of applications. One method to
purify a protein is through the introduction of an “affinity tag” that binds to a
chromatography medium/column. In this lab you will perform affinity chromatography
on His-tagged GFP and will subsequently assess the purity of your purified GFP.
Prelab Questions:
1. Do you think the method that we are using (His-tagged protein with Affinity
Chromatography) of purification would give higher or lower protein purity than
other methods of purification, such as Size Exclusion Chromatography or Anion
Exchange Chromatography? Why?
2. Using my calibration curve (given in the write-up), how much of your purified
protein product do you need to load onto the gel if your absorbance using the
Bradford Reagent at 595nm is 0.55 ? How much water should you add to equal
the correct final volume without loading buffer?
3. The Simply Blue stain that we use to stain the gel has a Coomassie Blue Stain that
stains the proteins blue. We use coomassie in another section of Lab 9 (it is
present in another solution). Can you think of what other solution / reagent
coomassie may be present in?

Large quantities of pure and homogeneous proteins are essential to a number of
applications, from scientific studies of protein function to pharmaceutical protein
production by the biotechnology industry. In the former example, impurities can
complicate or even invalidate the results of scientific experiments. In the latter, however,
contaminants in a pharmaceutical protein product can cause side effects that can
potentially endanger a patient.
As you learn in separations courses, compounds are separated and purified based
on differences in physical properties, and this same principle applies in biological
systems. Proteins can be purified on the basis of their size, solubility, density, charge, or
ability to bind to a compound. This lab will focus on the last of these approaches,
purification of a protein based on its affinity for a small compound, or ligand.
In a previous lab, you inserted the green fluorescent protein into a new vector
(pBAD). Similarly, the GSI’s have inserted GFP into a vector (pET19) that contains a
recombinant protein tag (His tag). In doing so, this tag has been fused onto the beginning
of GFP, so that when GFP is expressed from this vector, six histidine residues are added
to GFP’s amino terminus. This His tag binds to the metal nickel with high specificity and
affinity. Nickel immobilized on small beads is commercially available and can be loaded

Binding.onto a column. The protocol involves denaturing the protein sample by heating it in the presence of SDS and a reducing agent (DTT). The molecular weight of the polypeptide is inversely proportional to its mobility. whereas the reducing agent will reduce the intramolecular and intermolecular disulfide bonds. Remove the stopper and connect the column to the syringe (use the adapter provided). Collect 500uL fractions of flow through (this means that each 500 uL of flow through must be collected into a separate centrifuge tube and labeled). The second phase of this lab is to assess the purity of your sample through SDSPAGE electrophoresis. the band associated with the molecular weight of GFP will be the only band present on the gel. II. 4. You must wash and equilibrate the column (our column = 1 cm column volume). Wash the column with 3-5 column volumes (CV) of distilled water by slowly pushing water through the column. 1. Purifying and Eluting GFP 5. The molecular weight of our GFP is roughly 30 kDa and can be verified by a standard protein ladder of known MW species. If your sample is pure. 2. the gel is stained with a Coomassie Blue Stain to visualize the polypeptide bands. SDS will bind to the protein causing it to unfold. A crude solution containing GFP is then passed over the column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique for the separation of polypeptide subunits according to their molecular weight. and the GFP is stripped from the column for recovery by the addition of a histidine analog. After the electrophoresis is complete. The binding of SDS by the protein confers a net negative charge and the denatured polypeptide will migrate through a gel of known percent acrylamide in the presence of an applied electric field towards the positive electrode (anode). Try to push the water out at a steady rate by watching the volume in the syringe go down. . Preparing The Column You are given a syringe column packed with chelating sepharose precharged with bound Nickel. Procedures Protein Purification (Day One) I. 3. Practice maintaining constant flow by collecting 1 mL as your partner times you. to avoid introducing air into the system. Fill the syringe with distilled water. Equilibrate the column with at least 5 CV’s of binding buffer. Remove the snap-off end at the column outlet. the column is extensively washed. You may discard the water. Ideal flow rate is 1 mL/min. Apply cell lysate containing impure GFP (supplied by GSIs) to the column.

Read the absorbance of your binding and purifying fractions. Wash column with 5 CVs nanopure water and dispose of water flow through to clean the column before storage. You may use the UV source to help decide when you are finished loading. 10. 7. 8. Quantifying the Flow through and Amount of Purified Protein You must quantify the amount of protein that you have in order to know how much to load on a protein gel and to calculate a breakthrough curve. transfer each solution to their respective cuvettes. You will quantify all fractions collected (from binding. Add 20ul fraction sample to others. 11. Blank spectrophotometer with Blank2. When you are confident that all impurities are washed away. These will be used to calibrate the instrument. Label cuvettes and centrifuge tubes accordingly. You can do this using a Bradford Reagent which binds to the basic sidechains of amino acids and changes color upon binding. Collect 500 uL fractions of pure product. Add 20 ul binding buffer to Blank1 and 10uL elution buffer to Blank2.2M Imidazole). Blank / calibrate the spectrophotometer at 595nm with Blank1. 14. 7. 13. III. Wash away impurities using binding buffer. These should be your pure GFP samples. 9. Two blanks should be prepared with (1) binding buffer and (2) elution buffer. 12. The change in absorbance at 595nm detects this change. 9. . Set a timer for 10 minutes. Continue loading the column (and collecting fractions) until the flow through is a constant color of green (approximately 10 mL lysate). Collect 1 mL fractions of flow through for this step. 15. This indicates that you have reached the maximum binding capabilities of the column. and eluting steps). Flow 10-15 CVs buffer through the column to purify GFP. elute GFP from the column using elution buffer (0. 8.6. Place 780ul sterilized water in all centrifuge tubes. After 10 minutes. purifying. but continue eluting until you can no longer see GFP being removed from column. Read absorbance of eluted fractions. 5 CVs of elution buffer is usually sufficient. Add 200ul Bradford Reagent to each tube and mix the tubes thoroughly.

dilute your sample accordingly with sterilized water. Place centrifuge tube in the heating block for 2-3 minutes at 90oC. Remove from heating block. You will need this number when you run your protein gel. 2. II. Into gel box. This equation will give you the ug of protein in the cuvette. and will vary depending on technique of the experimentalist). You are now ready to load your sample into the gel. The total volume you have to work with is 15ul. Plug the absorbance that you obtained for your sample into the following equation to obtain a rough estimate of your protein content.0448 18. If absorbance is greater than 1. SDS-PAGE Electrophoresis (Day Two) I. Label with initials and concentration. prepare a second sample of that fraction using only 5 uL + 15 uL buffer to assure that measurement is accurate. Save the elution fraction containing the most protein. 4. (example: for a 1mg/ml sample you would add to your vial 5ul GFP product and 10ul water. Make sure this is well mixed. (this mixes well if you add the water last) . Calculate the amount of protein you will need to load onto the gel.16.) Your total volume should be 15ul. Make sure this is solution is mixed (can do this with your pipette). Preparing your sample 1. 19. Add 5ul loading buffer. Preparing the gel (This will be done by the GSI) 6. Divide that by 20ul (the amount that you put into the cuvette) and you obtain a concentration that should be representative of the concentration of your collected fraction. 3. add 80ml 10X running buffer to 720ml water. In a fresh centrifuge tube. 17.0. Record the concentration of this fraction in your lab notebook. (Absorbance at 595nm) = (ug protein present in cuvette)*0. 5. (note: the below equation is the GSI’s calibration curve. Ideally you want to load 5ug of protein. Place this fraction (your purified GFP product) in the refrigerator until we are ready to run our protein gel.

Wash twice with sterilized water. Release the sample into the lane SLOWLY so that it doesn’t flow out of the lane. 15. Remove protein gel from plastic and peel off the white tape allowing charge to reach the gel. Keeping the pipette in a vertical position. III. you may pipette water into it to wash it prior to loading your sample IV. Assemble gel box. pipette 18ul sample into your pipette. 21. Once sample is in the lane. Running / staining the gel (This will be done by the GSI) 16.7.Loading the gel 10. Place gel in gel box and remove the comb from the top. 17. it is better to load only 18ul into each lane. (prior to this. 12. When gel is finished running. Place the hat on the gel box and plug it into the voltage source. Using 1ul pipette tips. 9. remove the pipette very carefully. 14. Remove water from staining box and fill with ~100ml of Simply Blue. it is removed from the gel box and the plastic around the gel is cracked open exposing the actual gel. the gel is in a plastic cassette).5 hours. Gel will now stain for 1 hour on a shaker. 18. If you feel that another sample has contaminated your lane. 8. . 11. Be very careful that you don’t scratch the gel in any way! 13. To assure that no sample flows into nearby lanes. maintaining the vertical position. 19. Gel is run at 125V for 1. Each lane holds 20ul sample. Only 5ul of protein ladder is loaded. carefully insert tip into the gel lane that you wish to load into. Gently remove gel from plastic cassette and place it in a staining box. 20.

I recommend using Scion Image to obtain a plot. along with following information about the column. but to get an accurate % purity you must use a program which quantifies the color contrast on the gel. Gel can then be scanned on a scanner. How useful do you think GFP is as a reporter gene.scioncorp.22.htm 4. How do these plots compare to what you expected? 2. however. You can get a rough idea of the purity by eyeballing it. Guidelines for Analysis & Conclusions Section (Remember. For production of pharmaceutical proteins. create a dimensionless breakthrough curve. Dump out stain and fill staining box with water.) 1. Calculate the number of transfer units Nt.1g/cm3 3. The website to download Scion Image is: http://www. This section. For each set of fractions. Leave overnight to allow the gel to destain. need not be limited to these specific guidelines. Considering the chemical structure of histidine. GFP is often used as a “reporter gene. Use the SDS PAGE gel from this lab to quantify the purity of your pure GFP and of the unpurified GFP (which will be run on the gel as well by the GSI). considering issues of fluorescence quantification and tracking of fusion proteins? 6.” or a gene whose presence is easily detected or “reported” in cells. plot protein concentration vs. Scion Image will then compute for you the area under each curve which you can use to compute %purity. How easy do you think it would be to scale up affinity chromatography? . elution volume. Using the data from the binding step.com/pages/scion_image_windows. What is the rate limiting step of this chromatography process? avg particle diameter: 90 μm void fraction ε : 20% of column volume column volume : 1mL ρB = 1. these are points you should consider and include in your analysis. why would you think it would bind to nickel? When might it be detrimental to add a His-tag onto a protein? 5. large quantities of product are required.

D. Scopes.G. New York.K. (1994) Protein Purification: Principles and Practice. Voet. . Voet. John Wiley and Sons. R.. J. Springer-Verlag. (1990) Biochemistry.REFERENCES The QIAexpressionist: A handbook for high-level expression and purification of 6xHis-tagged proteins (1997) Qiagen. 824-829. New York. pp.