A Swift Introduction to Electrophoresis

Electrophoresis is a very common technology used in many different types of protein analysis, no matter if the protein of interest is purified or part of a complex sample. When optimizing conditions for expression of recombinant proteins, electrophoresis can be used to obtain information about protein yield at various conditions. Additionally electrophoresis can be used subsequently to purification by gel filtration, to verify protein purity and to confirm that the purified protein has correct molecular weight.

Electrophoresis of proteins is usually carried out by loading a sample into a well, to which a voltage is then applied; the varying size, shape and charge of molecules makes them move through the matrix at different velocities. At the end of the separation, the proteins are detectable as bands at different positions in the matrix.

Gel electrophoresis, as a means to separate proteins, is usually performed under denaturing conditions imparted by the presence of the detergent, SDS, both in the sample and as a constituent of the gel and running buffer. 1.4 g of SDS will bind to each gram of protein, so that any inherent charge on the protein is masked by the coating of negatively charged detergent micelles. Denaturing gels can be run under reducing conditions, where a reducing agent such as dithiothreitol (DTT) or β-mercaptoethanol is added to the sample buffer and heated. These reagents act by cleaving disulfide bonds between cysteine residues to disrupt the quaternary and tertiary structure of the proteins, creating linear chains of polypeptides. Proteins treated in this way migrate at rates that are a linear function of the logarithm of their molecular weights.

Alternatively, denaturing gels can be run under non-reducing conditions (no sample boiling and no added reducing agent) when it is important to maintain the native structure of proteins for further analysis.

Polyacrylamide gels, both as homogenous and as gradient, are the most commonly used matrices in for separation of proteins. In a complex sample where separation is desired over a wide range of molecular weights, a gradient gel with increasing gel density should be used. In such a gel, over a given time, small proteins will reach dense regions of the gel while larger proteins will migrate within less dense regions.

Hopefully that brief of electrophoresis was helpful but for more detail and information on its use please download our free handbook guide to protein purification or ask a question via the comments section below. Thanks for reading 

Getting Started on our DHFR Project

Time to get to our DHFR project. As we have explained before our aim is to express, purify and do some characterization of DHFR while learning a few things along the way.

In our previous posts, we discussed how to plan a project, identify the key characteristics of your sample and target protein, along with reviewing the key analysis methods to be aware of. We also looked at the thinking around whether you should tag your protein or not and looked at the key/common tags.

With that in mind, a couple of things we need to consider; since we are planning to characterize DHFR using biophysical methods, we are aiming for milligram levels of active protein at a purity of more than 95 %. For our purposes, it would make sense to add a small tag, such as the Histidine tag (unlikely to interfere with our analysis methods and would simplify the purification process). However, since we are doing this to learn and demonstrate how to use protein expression, purification and analysis methodologies and the large variety of tools and techniques, we have (deep breath :) taken the decision to express and purify human DHFR without a tag. Hopefully, this will not be too complicated.

Among the methodologies we plan to use, we have already discussed CIPP – Capture, Intermediate Purification, and Polishing. Another methodology that we plan to use throughout our project is Design of Experiments (DoE). DoE is a structured approach to experimental planning that provides a framework to explore parameters that may influence the outcome of your protocol. It will help you minimize the number of experiments that you have to carry out, while maximizing the information you get out of them in order to improve or optimize your desired outcomes. As it should add logic and structure, we will try to apply DoE to protein expression, purification and any other opportunities that we may come across as our project progresses.

In our experiment, the first step is expression. To express DHFR, we have chosen the most common host, E. coli. There are many different systems that can be used (and we will review the pros and cons of the most common in a future post) but we chose E. coli as it is simple, fast, reliable, low-cost, and easy to get high expression levels (remember we need 95%). One drawback with this host is that there are no post-translation modifications, such as glycosylation. With this choice of host, there is also a chance that we will get our protein expressed in inclusion bodies. While this would require having to refold the protein, it may not be a negative thing for the purification, since the inclusion bodies precipitate and therefore can be easily isolated, plus you get very high purity of your target protein if it is expressed in IBs. 

In our next post, we will look at the gene construct, vector and the cloning of the DHFR expression vector in the E. coli host. 

Meanwhile, if you are interested in learning more about Design of Experiments for protein expression and purification, you can take a look at our handbook.

When should I use a tag to purify my protein? (and when not?)

Sometimes we’re not particularly interested in purifying proteins. We just want to get it over and done with so as to get on with the experiments that will help us understand it role. This is where tagging your protein with something that adds biospecific affinity comes in handy. It allows you to simplify the purification protocol greatly, sometimes to the extent that you can use a standard protocol.

Remember though, tagging may not always be the right solution as adding a tag can introduce changes compared to the native protein, leading to undesirable effects. For example, if you are interested in drawing conclusions about function, having a tag may introduce uncertainty, or worse,  totally destroy or alters the function of your target protein. 

Also the tag itself can interfere with your ability to use the protein the way you want; important in using therapeutic proteins, where you want to be as close to the native form as possible. In most situations though tags, especially small ones like a His-tag, have no negative affects in terms of biophysical characterisation.

If you need a protein in native form

If you have decided that you need to have your protein in the native form, you can express it without a tag and go through the process of designing a protocol involving multiple chromatographic purification steps. It can be a little tedious and potentially unnecessary. Nowadays, with so many proteins already having been expressed, it is likely that you will be able to find existing scientific literature with information on your purified your protein, or an analogue.

As an alternative to expressing and purifying the native form, you can tag your protein and then remove it later. Whilst this simplifies the purification protocol, you’ll have to figure out what tag to use and how to remove it. You will also have to add the steps for cleaving the tag (manually removing it) to your protocol. It does not always have to be manual; there are systems such as AKTA pure that can automate this for you.

Removing the tag sometimes  isn’t very straightforward. It always includes using a sequence specific protease. The blood factors Thrombin & Factor Xa are most commonly used for this, but you need to make sure that their respective cleavage sequences (LVPR↓GS and IEGR↓) are not present in your target protein. Some tags, such as the Glutathione-S-Transferase (GST)-tag can be removed with a more specific proteases, such as PreScission protease (human rhinovirus 3C protease, cleavage sequence LEVLFQ↓GP), and this is extremely unlikely to effect your protein.

If you don’t need a protein in its native form

In addition to using a tag to simplify the purification process, there are actually a number of other benefits

• A tag can add the ability to use a generic detection method (such as a standard tag-specific western-blotting antibody, or an enzymatic assay in the case of the GST-tag)
• It may actually help overcome some challenges with your protein e.g. by stabilizing it or making it more soluble, as shown in this (very cool :) example of how a challenging spider silk protein was purified by using a special solubility tag in addition to a Histidine-tag
• Some tags can be fused to proteins for a broad range of other applications such as—labeling for imaging and localization studies, protein–protein interaction studies, and subcellular localization or transduction
• Tags also allow strong binding to chromatographic affinity media in the presence of denaturants which makes it possible to purify a protein that requires this, e.g. if it has been expressed in inclusion bodies. In this case, you can then also try to perform refolding of the protein while it is still bound to the column (too complex to describe here, we will discuss this in-depth in a future post..)

Pros	Cons
Using a tag
Simple, generic purification using affinity chromatography (AC) as a first step	Tag may interfere with protein structure and affect folding and biological activity
Tags are easy to detect, in comparison to the target protein, which allows for a generic detection method	If tag needs to be removed, cleavage may not always be achieved at 100 % and sometimes some amino acids may be left
Solubility and stability can be improved	Only some tags can be used under denaturing conditions
Purifying a native protein
Tag removal is not necessary	The purification and detection protocols need to be designed specifically
Purification can always be done under denaturing conditions	Problems with solubility and stability may be   difficult to overcome and may need special protocols to be developed

       

In summary, tag if you can – it simplifies your purification work massively, but don’t be afraid to express and purify your protein in its native form. 

Let us know your experiences working with tags, preferences and if you have any questions in the comments. Next time, we will look at the pros and cons of the most common tags used for protein purification.