Which agarose affinity resin should I use: 4B or 6B?

4b vs 6b resin

 

Choosing the right reagents is crucial when preparing to conduct affinity protein purification. After you’ve chosen a tag system to use, the next step is to choose an appropriate resin. This blog post will outline some of the differences between 4B and 6B agarose resin and how to choose the right resin for your needs.


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Agarose affinity resin


Affinity chromatography resin is composed of agarose beads. Agarose beads can be used in multiple protein purification methods including size exclusion and affinity chromatography. It’s worth noting that some suppliers call their agarose “sepharose”- this is just a trademarked name for the same base material. In order to purify a protein sample using affinity chromatography, a ligand molecule is coupled to the beads. When the crude lysate is washed over the column, the ligand captures the affinity-tagged protein and remains bound while other impurities are washed away.


Differences in agarose matrices


Agarose beads come in different levels of agarose concentration which impacts several qualities of the resin. A higher concentration of agarose means that more cross-linking will occur, providing a stronger and more stable resin. This means that the resin will be more resistant to pressure buildup and can tolerate a faster flow rate. Lower agarose concentrations result in a larger pore size, while higher concentrations have a smaller pore size (Figure 1). The number of crosslinks do not affect pore size (Figure 1). 


Figure 1. Agarose cross-linking and pore size at different concentrations. A- low agarose concentration, low crosslinking, B- high agarose concentration, medium crosslinking, C- high agarose concentration, high crosslinking. Source: “Introduction to Agarose Matrices,” Cube Biotech.


TriAltus Im7 affinity resins


TriAltus offers two varieties of Im7 resin: 4B and 6B. 4 and 6 refer to the % concentration of agarose in the resin sample. Both types of beads are 45-165 uM in size. 


4B Resin

6B Resin
  • Lower concentration of agarose
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  • Higher concentration of agarose
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  • Less cross-linking
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  • More cross linking
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  • Larger pore size
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  • Smaller pore size
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  • More sensitive to compression
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  • Stronger resin
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  • Slower flow rate 
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  • Faster flow rate
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  • 60 mg/mL binding capacity for Im7 resin
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  • 40 mg/mL binding capacity for Im7 resin
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    4B resin, because of its larger pore size, has a higher capacity of 60 mg/mL when tested with TriAltus’ 40 kDa model protein. 6B resin has about 40 mg/mL capacity. These capacity differences are preserved when a larger protein such as Cas9 (~160 kDa) is purified: 4B has 12 mg/mL capacity while 6B has 8 mg/mL. The tradeoff between the two comes down to capacity vs flow rate. Purifying large proteins with 4B resin may increase the yield due to the increased capacity and increased residence time due to slower flow rates.



    In summary, 4B resin has a higher binding capacity due to larger pore size, but requires a slower flow rate. 6B resin has a slightly lower binding capacity due to its higher agarose concentration, but is less susceptible to compression and can use a faster flow rate. When used as a part of the CL7/Im7 tag system, both resin options are able to purify challenging targets.

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