Resins & Protein Binding Capacities

Compare & Contrast 

When choosing what resin to use for purifying a protein, one of the most important characteristics to look at is protein binding capacity.  Protein binding capacity is a critical parameter for chromatography media because it determines how much media is needed in order to purify a certain amount of protein. 

This in term determines the column size needed, flow-rates of your chromatography system, and ultimately the total costs for purifying your protein. But, can capacity data reported from different vendors always be directly compared?

The short answer is "No" and in this post we will try to explain what protein binding capacity is, how it can be measured, and what to look out for when looking at your vendor’s specification sheets.

Firstly, the resin capacity for different proteins is often specified by suppliers based on

• different modes of measurements (dynamic or static)

• experimental conditions (pH, salt/conductivity, protein concentration)

• reference (capacity per milliliter wet resin or g dry resin)

Naturally, what protein has been used to determine the binding capacity is vital information in order to be able to compare specs. Unfortunately, in many cases, the method for determination of the binding capacity is not stated.

This makes it very difficult to compare the resins based on tabular values from the vendors.

What are the differences between static and dynamic capacity?

The static binding capacity (SBC, also called total protein capacity) is normally measured in batch mode in a beaker and is usually referred to as the maximum amount of protein bound to a chromatography medium at given solvent and protein concentration conditions. The size of SBC   varies significantly depending on the protein loaded. In these experiments, an excess of protein is loaded to give a maximum binding capacity. Protein loss is often over 50 %.

Dynamic binding capacity (DBC), on the other hand, is the binding capacity under operating conditions (i.e. in the packed affinity chromatography column during the sample application and washing procedures). The DBC of a chromatography medium is the amount of target protein that binds to the medium  under given flow conditions before a significant breakthrough of unbound protein occurs.

DBC is determined by loading a sample containing a known concentration of the target protein. The load of the protein sample on the column is monitored and will bind to the medium to a certain break point before unbound protein will flow through the column. From the breakthrough curve (see fig.1) at a loss of for example 10 % protein (named QB₁₀),  the DBC is found and the experiment is stopped. As this parameter reflects the impact of mass transfer limitations that may occur as flow rate is increased, it is much more useful in predicting real process performance than a simple determination of saturated or static capacity.

In most instruction manuals from GE Healthcare, you can find information on the DBC and how it has been determined.

How does GE determine its resin binding capacity?

GE Healthcare uses the DBC as the measurement for media packed in columns since it takes the flow rate and bed height of the column into consideration. It also takes into account ligand density, size of protein, and the porosity of the media which are the only factors included in the measurement of SBC.

SBC can be used to determine the binding/total capacity of bulk media.

However, as DBC is measured under operating conditions you will also get information on what the maximum load of your target protein to your column should be in order to avoid unnecessary loss.

If you have any questions please ask away via the comments section or for more detail check out our Protein Skills Handbook which covers this and many other aspects of protein science

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 

The Four Key Factors in Planning Protein Purification

When beginning a new project, where do you start? With a blank page, a bunch of hopes and good intentions? With a protein purification project whatever you do, before you begin, there are a couple of things you always need to consider.

What’s your ultimate goal?
This is the one thing you need to be crystal-clear about: what do you want to accomplish at the end of the project? Do you want to characterize your protein using biophysical methods? Determine the structure using X-ray crystallography or NMR? Use your protein in a functional study? Raise antibodies in a rat or a mouse model? Whatever the ultimate goal, it will decide the quantity of protein needed, the purity and analysis techniques used and these will have a major impact on your planning.

For example with our DHFR project, we’re performing a detailed biophysical characterization including activity and binding studies.

But with any protein purification, there are four key aspects to consider:

1.    Quantity
The quantity of protein needed varies enormously depending on goals. The scale can be from picograms (e.g. for mass spectrometry analysis) to kilograms (e.g. therapeutic proteins). The most common quantities that you will require within a research setting are in the µg to mg scale, for functional and structural studies.

2.    Purity
Again the level of purity required for a protein is intrinsically linked to your goals. To be able to raise antibodies, purities of 90-95% are enough. For crystallography or characterization studies, purity of 99% is often required.

3.    Activity
It is very important to decide whether the protein must be active after purification or not. Protein activity is not important in only a handful of applications; fundamental characterization studies, such as mass determination, amino-acid analysis, etc. Usually in today’s research, and as a rule of thumb, plan to retain the protein’s activity.

4.    Homogeneity
Size or charge homogeneity depends on questions like: is your protein likely to aggregate? What are the consequences for your ultimate goal if it does aggregate? A common pitfall is that a very pure protein sample could be in the form of inactive aggregates; determining if this is acceptable again hinges on the final application.

Fig: an overview of how the scope of a project should be set depending on application

Since our project includes assay development, we need to consider how much protein we have to purify before planning the protocol; so as to avoid low quantities of the protein in any further experiments. (From bitter experience :) many of us have learnt that an under estimate of how much protein you need, can lead to running out of protein half way through or before you have all the results you need. To avoid this, perhaps work in 10s of milligrams at least, as re-doing the experiment takes time and ultimately costs more than the increase in scale.) If you do run out, you may end up having to express and purify more protein and using different batches of purified proteins in the same experiments. This introduces another source of variance and uncertainty when you interpret your results.

So with our DHFR project we aim to purify 10mg quantities at a purity of >95 %, which would be more than enough to perform the activity and binding studies

A couple of tips when planning your project:

• Try to keep it simple and aim for the purity you need for your experiments, not above that

• Plan ahead, express and purify enough amount of protein from each batch to cover all experiments in the study

• Remember the more purification steps you use the more of the protein you will lose

Good planning will make the difference between a successful experiment and one filled with frustration and failure. 
In our next post we'll look at what you need to know about your sample, but in the meantime if you want to share your own tips or have any questions, let us know using the comments/contact boxes below. 

For more on how to simplify planning & execution of protein purification download our free handbook