It seems like everyone is purifying histidine-tagged (his-tagged) proteins, so how hard can it be? Affinity purification of these proteins is not difficult, but it is not foolproof. There are some simple steps that you can take before, during, and after purification to ensure that you get protein that will work for your application.
What you do before, during, and after affinity purification makes a difference
Before you start
Tip #1. Make sure you have good protein expression
Perhaps you received a clone from a colleague or are constructing one of your own. Regardless of the source, ensuring that your protein is being expressed at acceptable levels is essential before beginning purification.
Ideally, the protein of interest will be soluble. In E. coli, one way to determine whether the protein is soluble after cell lysis is to centrifuge the cells, recover the supernatant, and check both the pellet and the supernatant. Soluble protein will be in the supernatant. A common method for checking protein expression is SDS-PAGE analysis and Western blotting with an antibody to the protein of interest. Other analyses may be performed to determine protein functionality.
Always save an aliquot of the sample that you will load onto the purification column or chromatography medium. Keep it on ice or at 4°C.
Tip #2. Choose the right purification product
The goal of protein purification is to satisfy what the end application for the purified protein demands. Proactively choosing the right purification product can help you to achieve this goal. Factors to consider include the metal ion that will be used to charge the immobilized affinity chromatography (IMAC) medium, the sample source, and your format needs.
If your goal is to obtain very high purity protein in a single step, cobalt ions (Co2+) may be a better option than Ni2+. Samples such as eukaryotic cell culture supernatants can contain substances that cause stripping of metal ions from standard IMAC media. And if you do not have access to a chromatography system, select a manual format that will work for you.
During IMAC purification
Tip #3. Maintain the integrity of your protein
To minimize protein degradation, consider adding protease inhibitors to your sample after clarifying by centrifugation and filtration. Keep your starting sample on ice or at 4°C and work quickly. Another option to clarification is applying your unclarified sample to a column specifically designed for this purpose.
Think about adding reducing agents such as dithiothreitol (DTT) if protein oxidation is a concern. Follow the guidelines in the manufacturer’s instructions for the medium that you are using.
Maintain the integrity of your protein while bound to the IMAC medium by keeping all buffers and the medium ice cold or by working in a cold room or cabinet.
Tip #4. Save all flowthroughs
Save all of the flowthroughs from the binding and washing steps and keep them cold. It may be possible to recover your protein if it did not bind well to the chromatography medium or eluted in the wash. You may decide not to keep all the liquid. Minimally, save an aliquot of each flowthrough for analysis. If you plan to remove the his-tag after the elution step, save an aliquot of the sample before adding the protease.
After affinity purification
Tip #5. Perform a thorough analysis. Does the eluted protein meet your needs?
Analyze aliquots of samples from all purification steps using a method such as SDS-PAGE. A Western blot or other analysis may be performed to confirm the presence of the protein of interest. A functional assay is recommended if your application requires functional protein. If your protein is not sufficiently pure, consider optimizing the imidazole concentration in the washing steps, using a different metal ion during IMAC purification, or performing further purification using size exclusion chromatography (SEC) or another method.
You may want to know the multimeric state of your protein. Two options are to run a native gel or to perform analytical SEC. If your application requires isolation of monomers from aggregates, for example, preparative SEC may be used.
We recommend this article on recombinant protein production and purification as a general resource for beginners.
Which tip do you think is the most important? Let us know. Or send us a tip of your own.