Affinity purification is a frequently used technique for purifying proteins. During affinity purification, affinity tags or intrinsic properties of the proteins are used to bind to a ligand on agarose beads as a key step during the purification process.
Have you ever wondered how ligands get onto the agarose beads in the first place? If so, this is the article for you!
To conjugate affinity ligands, agarose beads are first “activated” by using chemistry to add a reactive moiety onto agarose. The added reactive groups then either bind directly to affinity ligands or to an intermediate molecule that will bind the affinity ligand.
In this article we’ll briefly cover the basics of affinity purification and discuss how affinity ligands are added to agarose beads.
Article Table of Contents:
Conjugating Ligands to Agarose Beads
This article is full of scientific terms that may not be so familiar. So in Table 1, we’ve laid out some of these terms and their meanings. Glance over them now, and as you go through the article, refer back to this table if you find yourself needing a recap.
In a recent article we covered how agarose beads are made – so if you’re interested in that, please check it out. Here, we’ll skip this information and get right into why you would want a ligand on an agarose bead, what types of ligands are used for affinity purification, and finally how those ligands are added to the beads.
Quick Article Vocab Guide
Table 1. Key Vocabulary Terms
|
Term |
Definition |
|
Affinity tag |
Peptide or protein tag used to purify other proteins |
|
Ligand |
Ion, small molecule, peptide, or protein that binds to an affinity tag |
|
Reactive Group |
Chemical moiety used to bind ligands to agarose |
|
Spacer |
Linker between the agarose bead and reactive group / ligand |
|
Activated |
Agarose is “activated” when a reactive group and spacer are chemically added to it |
Affinity Purification
Affinity purification is a common technique that leverages naturally occurring interactions to enable protein purification. Often, these interactions are facilitated by putting a “tag” onto the protein of interest. This tagging occurs during the cloning stage, and makes it so that a peptide sequence or a protein domain is present and binds to an immobilized affinity ligand.
While the tagged protein is bound to the immobilized ligand, other molecules flow through the column and are washed away. Then the bound molecule is eluted by introducing a molecule or buffer condition that disfavors the ligand-tag interaction (Figure 1).
Figure 1. Basic concept of affinity purification. A protein with an affinity tag is captured on beads with a complementary ligand, while other proteins flow through the column (middle). The tagged protein is then eluted by disrupting the interaction between ligand and tag (right).
Common examples of protein tags include his-tags, which are stretches of histidine amino acids that bind to nickel and other transition metals, and GST which is a protein domain that binds to glutathione. These interactions attract the tagged protein to agarose beads that have the corresponding ligand conjugated to them (Figure 1). See this article for more information about his-tags and other peptide affinity tags such as FLAG tags and strep tags, and this article for more information about GST and other solubility tags such as MBP and SUMO.
Before we move on, there is one more type of affinity purification we should talk about, and that is antibodies. Proteins A, G, and L bind to characteristic features of antibodies called heavy chains and light chains. Therefore, when these proteins are conjugated on agarose beads, they are used to purify certain types of antibodies (Figure 2). See these articles for more information about Protein A-, Protein G-, and Protein L-conjugated agarose beads.
Figure 2.
Protein G (green) binding to an antibody’s light chain (orange) and heavy chain
(purple) (PDB: 1IGC).
To summarize, there are a variety of affinity ligands that interact with protein or peptide tags for purification that include metal ions, small molecules, peptides, and proteins (Table 2).
Table 2. Affinity Purification Tag-Ligand Combinations.
|
Tag / Feature |
Ligand |
|
His-tag |
Ni2+, Co2+, Cu2+, Zn2+ |
|
Flag-tag |
Anti-Flag Antibodies |
|
Strep-tag |
Streptavidin |
|
Glutathione |
GST |
|
Maltose |
MBP |
|
SIM |
SUMO |
|
Antibodies – Heavy Chain |
Protein A |
|
Antibodies – Heavy Chain |
Protein G |
|
Antibodies – Kappa Light Chains |
Protein L |
Conjugating Ligands to Agarose Beads
Ok, so know that you know some of the common affinity tags and the ligands that they interact with, how do the ligands get added onto the agarose beads. In a word: chemistry! The ligands are added after the agarose beads have already been formed. If you would like to learn more about how agarose beads are made – check out this article.
To expand on the chemistry of attaching ligands to agarose beads a little, there are usually 3 components to the ligand (Figure 3):
- the ligand itself that binds to the peptide or protein tag
- the reactive chemical group that forms a covalent bond with the ligand
- a spacer that connects the reactive chemical group to the agarose bead
Figure 3.
Affinity ligand (blue) is conjugated to an agarose bead (gray) through a
reactive group (green) and spacer (gray line).
Another way of thinking about this is that agarose beads are customizable. By adding different ligands to the agarose beads you decide which type of protein or affinity tag the beads will capture. The different ligands need adaptors to attach to the beads, and these adaptors are the reactive group and spacers. And as we’ll discuss shortly, the specific type of adaptor depends on the type of ligand being added to the agarose bead.
We’ve already discussed the ligands themselves quite a bit, so check out the above referenced articles if you’d like more information about those.
Agarose beads are “activated” when a reactive chemical group has been covalently added that is now ready to bind to affinity ligands. Six main types of activated agarose for protein purification are:
- Cyanogen Bromide (CNBr) activated
- Amine activated
- Aldehyde activated
- Epoxy activated
- Maleimide activated
- NHS activated
The name tells you what kind of chemical group is active and ready to interact with the ligand (Figure 4). See Table 3 for the type of activation used to conjugate affinity ligands to many of the different types of agarose beads that GoldBio sells for protein purification.
Figure 4. Different activating groups (green) for covalently bonding to affinity ligands. The different types of groups are put onto the same bead for illustrative purposes. In actuality, an agarose bead would have many of the same types of reactive groups based on how the agarose bead was activated.
Table 3. Activation of GoldBio Agarose Beads
|
Agarose Bead Type |
Reactive Group |
|
Nickel and Cobalt |
Epoxy |
|
Proteins A, G, and L |
CNBr |
|
Streptavidin |
CNBr |
|
Glutathione |
Maleimide |
GoldBio’s nickel and cobalt agarose beads, for example, are first activated with epichlorohydrin, leaving an epoxy group that will bind to a Ni2+-chelating ligand, such as iminodiacetic acid (IDA). IDA binds to the activated agarose through the reactive epoxy group and chelates Ni2+ ions to interact with his-tagged proteins (Figure 5).
Figure 5. Ni
IDA Agarose. Agarose is activated (top right) creating an epoxy reactive group
to attach IDA which binds to Ni
2+. The Ni2+ ion serves as
the binding site for His-tagged proteins to the agarose beads.
As one more example, GoldBio’s Protein A, G, and L beads are conjugated to cyanogen bromide (CNBr) activated agarose beads. CNBr activation of agarose beads leaves a cyanate ester on agarose which binds to amine groups on proteins such as Protein A, G, and L (Figure 6).
Figure 6.
Protein G (blue) is conjugated to cyanogen bromide (green) activated agarose
beads (gray).
Lastly, as far as spacers are concerned, there are really two main considerations. One consideration is that the ligand needs to be far enough away from the agarose bead so that the affinity tag – ligand interaction can occur without the bead physically blocking this interaction. The other consideration is that the spacer needs to be chemically inert. Usually that means that the spacer is devoid of charge so that it doesn’t interact with proteins through a charge-based mechanism like what is intentionally used in ion exchange chromatography. Intermediate-length hydrophilic linkers are commonly used to satisfy all of these criteria.
So that’s how affinity ligands are added onto agarose beads. Check out the links throughout the article to other articles discussing affinity purification, and also check out the many types of agarose beads that GoldBio provides to enable affinity purification below.
