The reported binding capacity of GoldBio’s nickel agarose beads ranges from 6 to 80 milligrams (mg) of his-tagged protein per milliliter (mL) of resin. The exact binding capacity will depend on your purification setup, including your protein of interest’s size and purification buffers.

If you like to cook, then you know that the proportion of each ingredient in a recipe is really important. For example, if you’re marinating chicken and you quadruple the amount of chicken without increasing the seasoning, then you’re probably going to end up with some bland chicken.

It’s also important to keep relative quantities in mind when purifying his-tagged proteins with nickel agarose beads. Using the correct quantity of nickel beads for the amount of his-tagged protein you have helps to maximize the yield and purity of your purified protein, while also not wasting money using too many beads.

The reported binding capacity of GoldBio’s nickel agarose beads ranges from 6 to 80 milligrams (mg) of his-tagged protein per milliliter (mL) of resin. The exact binding capacity will depend on your purification setup, including your protein of interest’s size and purification buffers.

In this article, we’ll cover the binding capacities for different types of nickel agarose beads and how to estimate an appropriate amount of nickel resin to use based on your specific protein and purification protocol.

Article Table of Contents:

Different types of nickel agarose resin

What is a mL of nickel resin?

Binding buffer

How does the size of my protein impact binding capacity?

Related Resources

References

Different types of nickel agarose resin

GoldBio sells a variety of nickel agarose beads, each with different protein binding and buffer compatibility properties (Table 1).

Table 1. GoldBio Nickel Agarose Bead Binding Capacity

Nickel Agarose Bead Type	Binding Capacity	GoldBio Catalog #
Highest Density Nickel	80 mg/mL	H-390
Nickel NTA Magnetic	75 mg/mL	H-351
Nickel NTA HTC	60 mg/mL	H-355
Nickel NTA	50 mg/mL	H-350
Nickel Agarose Beads (High Density)	25 mg/mL	H-320
Nickel HTC	6 mg/mL	R-202

For example, if you want magnetic beads then Nickel NTA Magnetic Agarose Beads are your clear choice. For non-magnetic beads there are a variety of choices that differ by conjugating linker (NTA vs IDA), chemical stability, and binding capacity.

The binding capacity for these nickel beads ranges between 6 and 80 mg of his-tagged protein per mL of agarose bead resin.

What is a mL of nickel resin?

First, we want to start with understanding the context of a milliliter when it comes to binding capacity. So, let’s make sure we’re on the same page about what a mL of nickel resin is.

Most GoldBio nickel agarose resins come as a 50% (v:v) aqueous suspension in 20% ethanol. That means if you pipet out one mL of resuspended resin, there will only be about 0.5 mL of resin in there. So, you’ll want to pipet 2 mL of resuspended resin for each 1 mL of nickel resin that you’re using for your purification.

The math:

Resin = 0.5 * 1 mL aqueous suspension (AS)

Resin = 0.5 mL

We want 1 mL of our nickel resin …

1 mL resin = 0.5 * (AS)

1mL resin = 0.5 *(AS)

0.5 = 0.5

1 mL resin = 2 mL of aqueous suspension (AS)

What is resuspended resin? Resuspended resin just means that you want to invert the bottle of agarose resin a few times to make sure that all the beads are off the bottom of the bottle and in solution before pipetting some out of there.

All GoldBio nickel resins come as a 50% aqueous suspension, except for the Nickel NTA Magnetic Agarose Beads which are a 5% suspension (Table 2).

Table 2. GoldBio Nickel Agarose Bead Aqueous Suspension Percentage

Nickel Agarose Bead Type	Suspension	GoldBio Catalog #
Highest Density Nickel	50 %	H-390
Nickel NTA Magnetic	5 %	H-351
Nickel NTA HTC	50 %	H-355
Nickel NTA	50 %	H-350
Nickel Agarose Beads (High Density)	50 %	H-320
Nickel HTC	50 %	R-202

That means the math above is the same for all of the nickel beads except for the Nickel NTA Magnetic Agarose Beads. For the magnetic beads, you’ll need to add 10-times as much to get one mL of resin, which is 20 mL of aqueous suspension per one mL of resin.

Binding buffer

Your choice of binding buffer will influence the binding capacity of the beads, so if you are capturing substantially less than the reported binding capacity of your beads, and your protein isn’t really big, then it is worth considering if your binding buffer is limiting the capture efficiency of the beads.

We cover protein purification buffer compatibility with nickel agarose beads in more depth in this article, so check it out if you want more details. But, we’ll cover the highlights of those points here as well.

In GoldBio’s His-Tag Buffer Set and His-Tag Column Prep Protocol, we recommend using 50 mM Phosphate Buffer pH 8, 300 mM NaCl, and 10 mM Imidazole as the binding buffer.

Buffer pH is typically kept between pH 6 and 8, with pH 7-8 promoting maximal capture of your his-tagged protein of interest, and pH 6-7 enhancing the purity of your eluted protein.

Salt is helpful in preventing contaminating proteins from binding nonspecifically to the nickel agarose beads and to your his-tagged protein of interest. Typically, ~ 150 – 500 mM of NaCl is included in the binding and wash buffers.

Low concentrations of imidazole are useful for preventing contaminating proteins from sticking to the nickel agarose beads. Imidazole is the side-chain of histidine and competes for binding to the nickel ion. High concentrations ( > 250 mM) of imidazole are used in the elution buffer to evict your his-tagged protein of interest from the resin. However, lower concentrations (typically ~ 10-50 mM) of imidazole are used in the binding and wash buffers to prevent proteins with a few opportune histidine residues from binding to the nickel column (Hemdan et al., 1989; Salichs et al., 2009).

Denaturing agents such as urea (6 M) and guanidine HCl (8 M) are compatible with nickel agarose beads. This is a key advantage of nickel beads as his-tagged proteins that predominantly express into inclusion bodies can be isolated, denatured, and purified using nickel agarose beads.

Metal chelators, such as EDTA, and reducing agents, like DTT and TCEP HCl, should be limited as much as possible, or avoided completely. These are commonly included components in protein purification buffers, and are often required to prevent proteolytic cleavage and aggregation of your protein of interest. If you need to use these components, try to limit it below the concentrations listed in Table 3, then you can increase the concentration of EDTA or reducing agent as soon as you’ve eluted your protein off of the nickel column, if need be.

Table 3. GoldBio Nickel Agarose Bead EDTA and DTT Maximum Concentrations

Nickel Agarose Bead Type	Maximum [EDTA]	Maximum [DTT]
Highest Density Nickel	20 mM	20 mM
All Other Nickel Beads	1 mM	5 mM

How does the size of my protein impact binding capacity?

The bigger your his-tagged protein is, the lower the binding capacity of your column will be (Figure 1).

Figure 1. Tradeoff between protein size and nickel agarose binding capacity. The larger the his-tagged protein of interest is, the lower its binding capacity will be.

To think about why that is, let’s first consider an analogy. How many items could you catch in a baseball glove? That depends on what the item is, right? You could probably fit a dozen golf balls, three to four baseballs, two softballs, or one volleyball into the mitt. The larger the item, the fewer items will fit into the same space.

It’s a similar idea with binding to nickel agarose beads. There are many nickel ions conjugated to each agarose bead, so multiple his-tagged proteins can bind to the same bead (Figure 2).

Figure 2. With smaller his-tagged proteins, several proteins(green with Hs) can bind to nickel ions (blue circles) on the same agarose bead.

But larger his-tagged proteins means there is less space available, and fewer proteins can bind to each bead at the same time without running into one another (Figure 3).

Figure 3. Larger his-tagged protein (green with Hs) sterically block any others from binding to proximal nickel ions (blue circles).

Those are the factors that will influence the binding capacity of nickel agarose beads. If you need more details on how to calculate the exact amount of nickel beads that you should use, and how to determine if that amount of beads is the right amount, then check out this article where we cover those points in detail for glutathione agarose beads. Additionally, below we have links to GoldBio’s products that will enable you to purify his-tagged proteins in the lab, as well as some related resources to learn more and help troubleshoot.

Related Resources 

3 Small Peptide Tags for Affinity Protein Purification

What’s the Difference Between Nickel NTA and Nickel IDA Agarose Beads?

Affinity His-Tag Purification Protocol

His-Tag Column Prep Protocol

References

Hemdan, E. S., Zhao, Y. J., Sulkowski, E., & Porath, J. (1989). Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proceedings of the National Academy of Sciences of the United States of America, 86(6), 1811–1815. https://doi.org/10.1073/pnas.86.6.1811

Salichs, E., Ledda, A., Mularoni, L., Albà, M. M., & de la Luna, S. (2009). Genome-wide analysis of histidine repeats reveals their role in the localization of human proteins to the nuclear speckles compartment. PLoS genetics, 5(3), e1000397. https://doi.org/10.1371/journal.pgen.1000397

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binding capacity nickel agarose beads protein purification Simon Currie

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