How Much Resin for My Purification When Using Protein A, G or L? Binding Capacity Holds Answers

When affinity purifying a protein, one of the first questions you’ll ask yourself is “how much resin do I need for my purification?” As we’ll discuss, there usually isn’t an exact answer to this question, especially if you’re purifying this protein for the first time. But one of the things you’ll need to know that will help you approximate the desired amount of resin, is the binding capacity of the agarose beads that you’re using.

The binding capacity of agarose beads is just what it sounds like – it’s how much of a protein that particular form of agarose bead resin can bind. The focus of this article is on Protein A, Protein G, and Protein L agarose beads, so the relevant binding capacity is how many antibodies they can bind. But, the general concept of binding capacity holds for many other kinds of protein purifying techniques, as well.

The binding capacity of Protein A, Protein G, and Protein L agarose beads is in the range of 10-25 milligrams (mg) of human IgG per milliliters (mL) of resin. The exact binding capacity will depend on the antibody you are purifying and your buffer conditions.

This binding capacity is dependent on a few factors - most importantly:

• the type of antibody you’re trying to purify.
• the buffer you’re using for binding the antibody to the agarose beads.

In this article we’ll discuss those factors, how to estimate how much agarose bead resin you need, and how to adjust the amount you use for the next purification, if necessary, based on your results.

Article Table of Contents:

Binding Capacity

Antibody Type

Buffer Conditions

Estimating How Much Resin You Need

Interpreting the Binding Capacity of Your Experimental Setup

Additional References and Protocols

Related Products

References

Antibodies are frequently purified using Protein A, Protein G, or Protein L agarose beads. An important step in the purification process is binding the antibodies to the beads during the loading step (Figure 1)

Figure 1. Purification of antibodies. Antibodies bind to agarose beads conjugated with interacting partner molecules such as protein A, G, or L (column 3). After washing, antibodies are eluted with an acidic pH elution buffer that weakens the interaction between the antibody and protein A (column 4).

A key decision you’ll have to make early in the purification process is, “how much agarose bead resin do I need to purify my antibody?” If you’re doing batch chromatography using gravity columns, this generally means something in the range of 5 to 20 milliliters of the agarose bead resin. As we’ll discuss next, the binding capacity of the type of agarose bead you’re using will help you make this estimation.

Binding Capacity

The binding capacity of a particular type of agarose bead is how much protein that bead can bind to. It is usually reported in terms of mg of protein bound per mL of agarose bead resin.

As an example, earlier in the article, I mentioned that the binding capacity of Protein A, Protein G and Protein L is around 10-25 mg of IgG per mL of resin. So, another way to look at it is for every one mL of resin you use, proteins A, G or L will bind between 10-25 mg of your IgG antibody.

In Table 1 you’ll see the binding capacity for Protein A, Protein G, and Protein L agarose beads from GoldBio.

Table 1. Binding Capacity for Protein A, G, and L agarose beads

Since the binding capacity refers to mL of resin, it is important to know what this term means. Agarose beads are usually sold as a 50% slurry, meaning the solution is 50% agarose beads and 50% liquid solution. So, you’ll want to invert the bottle several times to make sure that the slurry is as homogenous as possible and there are not any big clumps of agarose beads stuck at the bottom of the bottle.

Then, you’ll pour twice the volume of desired resin into your plastic column. So, for example if you want 5 mL of agarose bead resin for your purification, you’ll pour 10 mL of well mixed slurry into your plastic column, then let the 5 mL of buffer run through leaving you with 5 mL of agarose bead resin (Figure 2).

Figure 2. The volume of the settled agarose resin (right) will be about half of the 50% slurry that you put into the column (left).

Antibody Type

You may have also noticed that the binding capacity is in terms of mg of Human IgG antibody. If you’re purifying a human IgG antibody, these binding capacities are super relevant for you – nice! But what if you’re purifying another type of antibody? You can adjust these binding capacities based on whether you think more or less of your protein will bind to these types of beads.

Protein A, Protein G, and Protein L are each different types of bacterial proteins with different binding affinities for different types of antibodies (Figure 3). For example, Protein A and G bind well to human IgG, but not to human IgA, IgD, or IgM antibodies. You would probably want to use Protein L agarose beads to purify human IgA, IgD, or IgM, but if for some reason you did use Protein A or G, the binding capacity for these different types of antibodies would be less than the numbers listed in Table 1.

Figure 3. Binding specificity of Protein A, G, and L to different types of antibodies.

The other thing that matters for binding capacity is the size of the protein that you’re trying to purify. With really small proteins, they can attach to each and every single binding site on the agarose beads. For really large proteins, even if there are additional binding sites available on the agarose beads, they will be blocked from accessing those sites by other, previously bound, large proteins (Figure 4),

Since most full-length antibodies are roughly the same size, this factor wouldn’t matter much as long as you’re purifying a full-length IgG, IgE, or IgD. However, if you’re purifying an IgM or IgA antibody, these are oligomers that are substantially larger and may have a reduced binding capacity. Conversely, if you are purifying a significantly smaller antibody fragment, such as a nanobody, then that would increase the binding capacity to the agarose beads as long as they still retain the epitope that binds to Protein A, G, or L (Dutta, 2018).

Buffer Conditions

The binding capacity of Protein A, G, and L agarose beads will depend on the buffer you are using to load antibodies onto the column. We discuss buffers in more detail here, but the key takeaway is that using an extreme pH, basic or acidic, or very high salinity buffers will reduce the binding capacity of the agarose beads. Using a binding buffer with neutral pH and lower salt, like PBS for example, is ideal for binding antibodies to the beads.

Estimating How Much Resin You Need

To estimate how much agarose resin you need, you’ll first need to estimate how much of your antibody you’re trying to purify. This will be a function of how well your antibody expresses, and how many cells are expressing the antibody. More conventionally, the latter term is just how large your expression culture is.

It is ok if you don’t know exactly how much antibody you are starting with. Typically, expression yields for antibodies are in the range of 10 to 1,000 milligrams of antibody per liter of culture (Robinson et al, 2015; Vink et al, 2014). This may seem like a wide range, but in practice the yield depends upon the exact antibody you’re expressing, what kind of expression system you’re using (mammalian vs. bacterial cells), and finally the exact details of your expression protocol.

For the purpose of this article, let’s assume you are starting with 100 mg of your antibody, and using Protein A agarose beads for purification. According to Table 1, you would want to use 4 mL of Protein A resin to purify your antibody, which I calculated by dividing the 100 mg of antibody by 25 mg/mL binding capacity of the resin. Recall that the agarose beads come as a 50% slurry, meaning that you would want to pour out 8 mL of well-mixed slurry to yield 4 mL of Protein A agarose bead resin.

Interpreting the Binding Capacity of Your Experimental Setup

Your estimates of how much antibody you’re starting with and how much agarose bead resin to use likely won’t be perfect, but in most cases, they will probably be good enough to purify some protein for you to work with. And you can always analyze your purification to determine if you should use more or less agarose resin for a subsequent purification.

To analyze your purification, you’ll want to run an SDS-PAGE gel. We discuss SDS-PAGE gels generally here, so check it out if you want more background about the type of analyses we’re discussing below.

For the first hypothetical example, let’s consider what your gel would look like if you estimated the correct amount of agarose resin. Most of your protein will be in the elution fraction, and this fraction will be pretty pure, meaning there is mostly just your protein and very little of other proteins (Figure 4). You may still see a little of your protein in the flow-through, and that’s ok because as we’ll discuss below, having none of your protein in the flow-through can be a problem.

Figure 4. Example of a hypothetical SDS-PAGE gel for antibody purification with Protein A, G, or L agarose beads. The antibody is in green whereas contaminating proteins are in blue. Approximately the right amount of agarose beads was used as most of the antibody is in the elution fraction with good purity.

Ok, now let’s go through what the gel would look like if you underestimated the amount of agarose resin needed for your purification. Again, you will have your antibody in the elution with good purity. However, you will also have a lot of your antibody in the flow-through lane as well (Figure 5). This tells you that next time you purify this protein, you should increase the amount of resin used to capture, and elute, more of the antibody.

Figure 5. Example of a hypothetical SDS-PAGE gel for antibody purification with Protein A, G, or L agarose beads. The antibody is in green whereas contaminating proteins are in blue. Too few agarose beads were used and a lot of the antibody is in the flow-through fraction.

Lastly, let’s consider a situation where you used too much agarose resin. In this case, your protein will be in the elution fraction and there will be none, or very little, of your antibody in the flow-through. However, your eluted antibody may be less pure – meaning there are other proteins also in the elution – because these other proteins bound nonspecifically to the Protein A, G, or L agarose beads (Figure 6). You can optimize your wash buffer to try to separate these undesired proteins from your antibody, but often the easier step is just to reduce the amount of agarose bead resin used to reduce the availability of non-specific binding to the beads.

Figure 6. Example of a hypothetical SDS-PAGE gel for antibody purification with Protein A, G, or L agarose beads. The antibody is in green whereas contaminating proteins are in blue. Too many agarose beads were used as all of the antibody is in the elution and there are contaminating proteins that coeluted through nonspecific interactions with the agarose beads.

Remember, protein purification is often an iterative process! Even if you didn’t estimate well your first time, you can always learn from that using this type of analysis to make the second go around even better.

So that’s all about the binding capacity of Protein A, Protein G, and Protein L agarose beads, what it depends on, and how to use that information when purifying antibodies. For more information, check out the links to our related articles and relevant products below and throughout this article.