How can you teach strategies for protein purification without a lab?
Protein Purification is an award-winning program which has been widely used in schools, colleges and universities since 1983. It guides students (and experts!) through some of the more commonly-used protein separation techniques and lets them experiment. Students can begin with a simple mixture of proteins and follow a tutorial to see how these behave during gel filtration and ion-exchange chromatography. Afterwards, students can go on to design and test full purification protocols using more complex mixtures of proteins. The behavior of proteins in the mixtures is based on real proteins.
The program assumes that users are familiar with the theoretical background to the most common separation techniques, enzyme assays etc. and that they understand the concept of the isoelectric point of proteins. The isoelectric point is the pH where the net charge of a protein equals zero.
The program is very complete. You use several types of chromatography: ammonium sulfate fractionation, heat treatment, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography.
Handbooks on chromatography methods - These are based on the Pharmacia chromatography handbooks. Currently, you can download these for free from a company called Cytiva. The handbooks cover Size exclusion chromatography (also known as gel filtration), Ion Exchange Chromatography (Anion and Cation methods), Western blotting, Hydrophobic and Reversed Phase Chromatography, Affinity Chromatography for Antibodies, Tagged Proteins, and Specific Biomolecules.
1. I picked the Easy 3 mixuture and decided to purify protein #2. I am starting with 30 mg of protein and 2000 units of activity.
2. I chose to look at my mixture by using 1d and 2d polyacrylamide gel electrophoresis (PAGE) and an immunoblot.
Data from the 1d gel and the immunoblot show me that my protein has a molecular weight around 20 kilodaltons.
3. I looked at the results from a 2d PAGE also to find the isoelectric point.
The 2d immunoblot shows me that my 20K protein has an isoelectric point around a pH of 7.3. I see there is a 40k protein with a similar isoelectric point and a protein that's the same size as mine with an isoelectric point closer to a pH of 6.
4. Since my protein is small, I'm going to try gel filtration to see if I can remove the larger protein. I click Hide Blot, click Separation, then I click one of the gel filtration resins and read the information to see which one will exclude proteins around 40k. I'm doing this because I would like the bigger protein to flow through the column and my protein to come out later. After reading the info and looking at the fractionation ranges for different resins, I chose Sephadex G-50 and clicked OK.
My elution profile is below.
My protein and another 20K protein are smaller, so they should come off the column later and be in the second peak.
5. I clicked Fractions and chose Assay enzyme activity to confirm this.
6. The enzyme assay shows my enzyme is in the second peak. Now, I'm going to pool the fractions that contain this activity. I pick fractions 32 through 40.
I click Fractions > Pool fractions and enter the numbers. I clicked Check to see make sure I have the right ones.
After checking, I decided to use fraction 41 also since it contains enzyme activity.
Then I click OK.
7. Now I want to remove the other 20k protein, since the isoelectric point is different, I'm going to try Ion exchange chromatography. I click Separation and choose Ion exchange chromatography. Proteins should have a negative charge if the pH is above the isoelectric point. I chose DEAE cellulose and a salt gradient. At pH 7, my protein will have a positive charge and should flow through the column, so I'm leaving the pH at 7 and I click OK.
I left the gradient at the default values and clicked OK.
These results look pretty good. My protein flowed through the column and the other protein stuck. My last steps are to pool my fractions and run a 2d gel and make sure that my protein is pure.