- 3 Examine The Chromatogram For Q Sepharose Run At Ph 10 Specifically The One That Employed A 0 0 5 M Nacl Gradient At 1 (99.46 KiB) Viewed 16 times
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A 280nm 2.0 1.0 0 10 20 Q-Sepharose @ pH 10.0, 0-0.5 M Salt Ion Exchange Chromatography on Q-Sepharose 80 90 50 60 70 Fraction Number 30 40 100 110 120 266 Enzyme Activity (Units/ fraction)
280nm 2.0 1.0 0 10 20 Q-Sepharose @ pH 10.0, 0-1 M Salt Ion Exchange Chromatography on Q-Sepharose 50 70 80 60 Fraction Number 30 40 90 100 110 120 266 Enzyme Activity (Units/ fraction) 0
A 280nm 2.0 1.0 0 10 20 ÞEAE-cellulose @ pH 10.0, 0-0.5 M Salt Ion Exchange Chromatography on DEAE-cellulose 50 70 80 60 Fraction Number 30 40 90 100 110 120 266 Enzyme Activity (Units/ fraction) 0
2.0 A 280nm 1.0 0 10 20 DEAE-cellulose @ pH 8.0, 0-0.5 M Salt Ion Exchange Chromatography on DEAE-cellulose M 50 60 70 Fraction Number 30 40 80 90 100 110 120 0.5 [salt] (molar) 0
3. Examine the chromatogram for Q- Sepharose run at pH 10, specifically the one that employed a 0-0.5 M NaCl gradient (Panel D). At what [salt] did Protein 1 elute? Explain the basis for the observation. 4. Examine the chromatogram for Q- Sepharose run at pH 10, specifically the one that employed a 0-1 M NaCl gradient (Panel E). At what [salt] did Protein 1 elute? How does this result compare to the 0-0.5 M NaCl gradient (Panel D)? Explain the basis for any observed differences. 5. Compare the chromatograms for Q- Sepharose (Panel D) and DEAE-cellulose (Panel F) run at pH 10. How do they compare? Explain the basis for any observed differences. Students are required to consider a situatio where Protein 1 elution did not occur and think about why this happened. Students discover that a strong Protein 1- column interaction can be overcome by increasing the final [salt] of the linear gradient. The comparison illustrates difference between strong (Q-Sepharose) and weak (DEAE-cellulose) anion exchangers. An effective answer will require students to consider the structure of the ion exchange groups on each resin.
6. Examine the chromatogram for DEAE- cellulose run a pH of 10 (Panel F). How might the results have changed if the equilibration pH was lowered to pH 8? Try it with the program and explain the basis for the observed result. Students will discover that Protein 1 does not adsorb to DEAE-cellulose at pH 10, but does at pH 8. An explanation will require students to think about the structure of the DEAE-cellulose ion exchange group, how its charge is affected by pH, and how this relates to Protein 1 adsorption.
Notes: • Q Sepharose is a resin with a quaternised nitrogen (i.e. a nitrogen with 4 groups attached and a positive charge). As it has a permanent positive charge, Q Sepharose remains an anion exchanger at all pH values. You still need to equilibrate at a chosen pH, as this affects the charge on the proteins in the mixture being purified, depending on their pl. I Set the gradient limits from 0.0 to 0.5 M salt. Click OK. o This defines the conditions that are used to elute the proteins from the anion exchange resin. A chromatogram will appear. Notes: . The chromatogramme shows the absorbance measured at 280 nm. Proteins will absorb UV light at this wavelength, so this is a way of showing which fractions off the column have protein in them. • You should remember from watching the video on ion exchange chromatography that they had different fractions in tubes that they labelled. In the video there were only a few tubes. For many protein purification applications it is common to have a lot of tubes, this simulation has many! • Proteins that did not adsorb to the column are those that eluted before the start of the linear salt gradient; these proteins emerged in the "flow through" (0 mM salt). The salt gradient is shown as a pink line, and you will see the gradient only starts after about fraction 32. ▸ Go to: Fractions>Assay enzyme activity. If Protein 1 eluted from the column a red trace will indicate where.
Notes: • When doing protein purification, it is usual to obtain some measure of the activity of your protein of interest, so that you know which of the protein-containing fractions identified by the A280 measurement (absorbance at 280 nm) had your protein of interest in it APPENDIX - Understanding the output from Andrew Booth's Software An example elution profile Methods Elution profile lon exchange chromatography on Q-Sepharose 4.0- Red peak is the enzyme activity, shown after you run the (virtual) assay. It shows you where your protein of interest is. Just because a protein peak coincides with the activity peak, doesn't mean that the only protein present (represented by the blue peak) is your protein of interest, there may be others there, too. Use the gels of pooled fractions to check that. Absorbance at 280 nm Unbound proteins elute in the washes before the NaCl is applied 2.0- 17 ton Blue peak shows the absorbance at 280 nm, representing where any proteins are present. 40 Pink line is the NaCl gradient Start of NaCl gradient at this fraction 50 60 70 80 Fraction number Ignore this number, it is an arbitrary value of the enzyme activity from the assay. We do not use it in this exercise. 533 If you had a protein eluting in fraction 85 you estimate the [NaCl] from the ratio of height of the line above fraction 85 to the height of the line at the end. If you salt gradient went to 500 mM (0.5 M), then [NaCl] =500 (height at F85)/(height at end) 90 100 110 120 0 Enzyme activity (Units/fraction)
The plot has fraction number on the x-axis. As the proteins are separated on a column, the liquid that flows through (elutes) is collected, typically a few mL at a time in separate tubes. Each tube is a "fraction" of the total eluate. The first few fractions collected are just collectin the liquid that was stored in the column, followed by fractions that are washing off unbound material. It takes time for anything applied to the top of the column to flow through to the bottom. Here we are interested in the proteins in the mixture applied to the top. The blue line represents the absorbance at 280 nm. This is a wavelength used to indicate the presence of protein - some aromatic amino acids absorb light around that wavelength. This shows us where each protein from the mixture elutes. In the picture above there is peak for proteins that elutes around fraction 20. This/they didn't bind to the column at all, and just flowed straight through. In this case, Q-sepharose is usually positively charged at the pH of a experiment and so binds negatively charged proteins. That means this first protein (or set of proteins), which does not bind, must be either positive or neutral at the pH of the experiment setup. Anything that elutes after the start of the salt gradient must have bound to the column and needs a salt to elute (see notes on pink line below). The red line represents results for a specific assay for the protein on interest. This will give a response only for the protein you are trying to purify. We can see that the response is only in the flow-through (around fraction 20), which means the target protein does not bind to the column in this case. The pink line represents the salt gradient. Hopefully from the pre-study you have picked up he idea that you can elute proteins from an ion exchange column by providing a high concentration of a competing ion. NaCl does this really well, and your experiment will have defined the salt gradient, maybe starting with none and moving up to say 500 mM. The first ractions are collecting what does not bind, and to ensure that the non-binding proteins are
separated from binding ones, there is usually a wash with a buffer without a high salt concentration. Here the first 32 or so fractions are wash, and then the salt concentration rises linearly towards the maximum at the right hand side. You can estimate the salt concentration where your protein of interests elutes from the pink line - easy here, as it elutes before the salt gradient starts. Later on, you can measure the height of the pink line on the right (say in mm) and you know the salt concentration (maybe 500 mM, depending on your setup). By dividing 500/height you have the number of mM/(mm height gain of pink line). Then measure the height of the pink line where your protein elutes to estimate the mM of NaCl in the elution.