Introduction to agarose beads for protein purification

Agarose matrices have been used for protein purification for decades, and a broad variety of agarose-based matrices is commercially available. Physical properties can vary significantly between the different matrices. This short summary shall support you in finding the optimal product for your research.

 

Superflow, Sepharose, Agarose - what's the difference?

In the past 20+ years, many developments have been made, and names have been created for different kinds of agarose matrices:

 

     

 

Sepharose   

GE trademark for crosslinked agarose in different concentrations and sizes. Properties of Sepharose vary depending on agarose concentration and degree of cross-linking, and not all Sepharose matrices are suitable for applications such as FPLC.

 

            
  Superflow   

Offered by several companies. The term describes a variant of agarose that is highly cross-linked and pressure-stable, thereby suitable for FPLC experiments. This term is mostly used to distinguish pressure-stable agaroses from others that show a lower degree of cross-linking and are mostly used for batch purifications only.

 

 
 

Cube Biotech   

PureCube Agarose   

PureCube 100 Agarose   

Our PureCube Agarose is highly-crosslinked and pressure-stable and therefore comparable to Sepharose and Superflow matrices (see Table 1). For high flow rates and uncompromised protein binding capacity, we recommend our novel PureCube 100 Agarose, offered for His affinity purification.

 

 
 
 
   

Cube Biotech

   Supplier G "Sepharose"   Supplier Q "Superflow"
Material
  Highly cross-linked agarose   Highly cross-linked agarose   Highly cross-linked agarose
Agarose concentration
 
  PureCube: 7.5%
  PureCube 100: 6%
  On request:
4%/ 5% /9%
 

6% / 4%

  6%
Pore size
  medium   medium   medium
Mean particle size
 

 PureCube:

40 µm

 PureCube 100:

50-150 µm

 PureCube XL:

300-500 µm
 

34 µm (HP)

90 µm (FF)

  60-160 µm
Recommended
linear flow rate
 

>500 cm/h (PureCube)

>1,000 cm/h (PureCube 100)

 

>150 cm/h

  >150 cm/h
Max. flow rate
  6 ml/min  

4 or 20 ml/min

(1 or 5 ml column)

  20 ml/min
 
Binding capacity:
His-tagged proteins and Ni-NTA

 

  up to 80 mg  

40 mg

 

up to 50 mg

 
 
Table.1: Comparison of three commercially available agarose matrices.
 

 

 
 
Crosslinked Agarose chemical structure   Agarose scanning electron micrograph  
 
 
Fig. 1: Chemical structure of crosslinked agarose.
Fig. 2: Cube Biotech Agarose beads, stained with bis-ANS dye. Kindly provided by PAIA Biotech, Cologne, Germany
 
Fig. 3: Scanning electron micrograph of crosslinked agarose at 50,000 x magnification.
Kindly provided by Anders S. Medin, PhD Thesis, Uppsala University 1995
 
 

 

Agarose concentration and crosslinking

The basic material of agarose beads is - obviously- agarose, a linear sugar polymer. This polymer is chemically crosslinked (see figure 4) to provide thermal and mechanical stability. Concentration of agarose strongly influences a range of properties, including

 

  Pore size  

The higher the initial agarose concentration, the smaller the pore sizes that form after crosslinking. This is particularly important if the agarose is to be used for size exclusion chromatography.

 

 
Pressure stability  

The higher the initial agarose concentration, the higher the degree of crosslinking, and the more pressure-resistant the beads become.

 

 
 
 
Fig. 4: Influence of agarose concentration and degree of crosslinking on pore size and rigidity of the matrix.
 
A: low concentrated agarose
B: high concentrated agarose with low degree of crosslinking
C: high concentrated agarose with high degree of crosslinking.
 
Green lines: Agarose polymer, Red dots: Crosslinking events
 

Surface chemistry

Agarose itself is a neutral molecule that shows very low unspecific binding to proteins. For use in protein purification, it can be easily modified with a range of surface chemistries, for example

 

Affinity chromatography  

Ligands such as NTA, glutathione, or antibodies can be covalently attached to agarose surfaces, to enable purification of proteins via a specific interaction.

 

 
  Ion exchange chromatography  

Functional groups such as quarternary amines, DEAE, sulfopropyl or carboxymethyl can be coupled to the agarose, leading to strong or weak anione exchangers, or strong or weak catione exchangers, respectively. For ion exchange chromatography, proteins do not require an affinity tag, making the method suitable for the purification of proteins from natural sources.

 

 
  Gel filtration / Size exclusion    chromatography   

Crosslinked agaroses can be used for size-exclusion chromatography without further modification. Resolution and size range depend on pore sizes. As a rule of thumb, the correlation of agarose concentration to separation capability is as follows:

 

Agarose concentration

 

Protein exclusion limit

(globular proteins)

4%   ca. 30,000 kDa

5%

 

ca. 10,000 kDa

6%

 

ca. 4,000 kDa

7.5%

 

ca. 1,200 kDa

9%   ca. 150 kDa
 
 

 

Table.2: Relationship of agarose concentration to protein exclusion limits.

 

 

 

 
 
 
 
 
 
Fig. 5: PureCube Agarose shows a higher dynamic binding capacity than a competitor matrix with 90 µm bead size.
 
BSA was purified via two ion exchange matrices in a Tris buffer at pH 7.4 in a set of experiments done at flow rates from 25 to 500 cm/h, corresponding to 0.3-4.0 ml/min in a 0.8x2.5 cm column. Protein yields obtained with the competitor matrix significantly decreased when flow rates higher than 100 cm/h (0.85 ml/min) were applied, whereas PureCube Agarose matrix showed reproducibly high protein yields even at high flow rates like 500 cm/h (4 ml/min). Data kindly provided by BioWorks, Sweden.
 

Size

Agaroses come in different sizes, and size distributions. These have an impact on the physical properties of the purification matrix.

 

     Pressure  

The smaller the beads, and the more narrow the size distribution, the higher the pressure-resistance of the beads. On the other hand, the larger the beads, the faster the flow rate in batch and FPLC experiments.

 

 

 

Binding capacity  

The smaller the beads, the higher the ratio of surface to volume, and the higher the binding capacity of the beads. However, note that the binding capacity also depends on other factors such as type and size of ligand, and density of ligand coupling.

 

 
  Dynamic binding capacity  

The binding capacity of an affinity chromatography material  decreases when pressure builds up inside the column at higher flow rates. This effect depends on agarose bead size, agarose content, and degree of crosslinking. Dynamic binding capacity tells us if an agarose material can be used at higher flow rates without influencing protein yields in a purification.

See Fig. 5 for a comparison of PureCube Agarose and competitor material.