Purification of a tagged protein may seem like a pretty routine task. You express your protein of interest, lyse the cells, put the lysate through the resin, elute, and your freshly purified protein is ready for the world. Unfortunately though, this process can have issues that may leave a researcher banging their head against a wall.

Troubleshooting purification of a tagged protein

Maybe your protein of interest just washed through the column without binding, or came off in one of the wash steps. Maybe there wasn’t as much in the final elution as you had hoped or the final concentration is too low to easily work with.

There are a lot of things that can cause issues for your purification, and this post will go over some of the most common causes and some of the best troubleshooting solutions.

1. No protein is present in the eluate

You went all the way through your purification, and there's no protein in your final eluate. Don't panic! This problem can occur for a few reasons.

The first reason could be that your protein of interest isn’t correctly expressing the affinity tag. To check for this, you should have your DNA construct sequenced from the promoter through the affinity tag to make sure there were no cloning errors (especially if a portion of the insert came from a PCR fragment) and that the protein coding region is in frame.

By making sure the sequence is verified, you can be sure there aren’t any internal translation start sites to interfere with your N-terminal tag, or any premature stop codons affecting your C-terminal tag.

The second reason may be that you’re not adding a sufficient amount of protein to your affinity resin. A good way to check your protein's expression level before purification is to run a fraction of your crude lysate on an SDS-PAGE gel, blot to a membrane, and perform a western analysis with a primary antibody targeting your affinity tag.

There are many antibodies to a wide variety of tags commercially available, and this is a good way to determine expression level of a construct. Along with showing that your protein of interest is being expressed, it can also show if your protein is being degraded during cell lysis.

The gel and western blot can also help optimize your induction conditions (i.e. differences between potential hosts, what concentration of inducer, how long to induce, at what temperature, etc).

By being able to compare end protein levels before attempting purification, you can make sure that you’re starting off with a large volume of target protein, which you can use to better match your resins capacity. Once you know that your construct is correct and your protein is expressing correctly, you can move on to optimizing the binding and washing conditions.

2. Potential issues with binding and washing

The washing procedure can be a two way street. If your washing conditions are too stringent, you run the risk of removing your protein off the bead before elution.

If they aren’t stringent enough, you risk contaminating your purified protein with other cell proteins that weakly bind to the resin.

If your protein of interest is being removed from the resin during one of the wash steps, this indicates that either the protein isn’t binding as well to the beads as it should be, or that your wash conditions are too stringent.

A possible cause of poor binding could be that your affinity tag isn’t accessible, and therefore isn’t binding to the resin well. If this is the case, you may need to run your purification under denaturing conditions to make sure the tag is fully exposed.

If your wash conditions are too stringent you may need to lower the buffer concentration and check the pH of the buffer.

If your wash conditions aren't stringent enough, you may need to run a buffer gradient to determine which concentration and pH gives you optimal performance.

3. Final elution issues

If after you’ve made sure that your protein is being properly expressed, and the wash conditions are just right, it’s time to elute. Hopefully this step will go pretty smoothly, but you could face the same issues as the washing step.

If your elution conditions are too mild, you risk leaving a significant amount of protein on the beads, but if the conditions are too harsh you may have to take extra steps to remove any contaminants that may also be eluted.

You can try different pH’s and concentrations of your elution buffer to try and alleviate these issue with a single purification. Just make sure any elution buffer you use will be compatible with your downstream applications, although there are always techniques to help clean up your eluted protein and get it ready for its next step.

If however, you’re still getting a large quantity of additional cellular protein purifying with your protein of interest, you may want to consider an additional purification step with a fresh resin. This will hopefully give you the purest final product, with only some loss of total protein.

Hopefully this information will set you on the right path in your protein research, and if you still have any questions there are a lot of really good resources both online and in print. Here at GoldBio, we want to make sure you are getting the most of out of the products you get from us, so please contact our technical support staff with any questions or issues.