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Affinity Purification

Affinity purification (also called affinity chromatography) is a technique in which the difference in absorption depends on the specific affinity between a substance fixed in the separation material (the absorbent) and the desired component in the mixture (the ligand). It is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures. It is usually performed in columns.

Expedeon offers a range of products offers cost-effective solutions that offer unparalleled performance and efficiency.

Principle:

For affinity purification to be successful, a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand is required. This helps to determine the selection of an appropriate affinity ligand and purification procedure.

Method:

The process is extremely simple. The ligand is attached to a solid support to form a specific adsorbent to which the biological system, usually in the form of a homogenate or extract containing the molecular species of interest, is exposed. Only the molecular species that shows appreciable affinity for the ligand will be retained or retarded; other materials which have no specificity for the insolubilized ligand will pass through the column. The molecular species that have become attached to the adsorbent must then be eluted without destroying its biological properties.

Table below shows which of our products can be used depending on the type of recombinant protein you need to purify:

Type of recombinant proteinResin/bead/matrix
His6-taggedNi NTA (mainly) or Cobalt NTA
GST-taggedGlutathione
AntibodyProtein A/ Protein G/ Protein L/ Protein A/G
If Ab is for conjugation
AbSelect and AbPure products
Ligand binderAmintraTM NHS activated (need aminated ligand)
Some plasma proteinsUFH
MBP-tagged proteinsMBP Resin (maltose)
ATPases, KinasesATP resin

GTPases
GTP resin

ATPase & GTPase assay Kits 

Expedeon’s PiColorLock™ phosphate detection reagent changes color in the presence of inorganic phosphate (Pi). The unique formulation of PiColorLock™

  • Colorimetric assay
  • Compatible with almost any assay buffer
  • Stable reagent formulation
  • Unique accelerator
  • Unique stabilizer
  • Wide linear range
  • The PiColorLock™ – Pi complex is very stable

Graph showing the PiColorLock™ stabilization of the substrate of the enzyme in unstable in acid.

 

Figure 1. There are very significant additional benefits of PiColorLock™ systems if the substrate of the enzyme is unstable in acid. The terminal phosphate in nucleoside triphosphates is particularly prone to non-enzymatic hydrolysis, and further hydrolysis of diphosphates (e.g. ADP, GDP) to their corresponding monophosphates also readily occurs in acidic environments.

Affinity Resins – Find out more below or browse our full range

PiBind™ Resin
Provides a quick and easy way to remove contaminating Pi from buffers. The resin works over a broad range of pH values and is unaffected by many commonly used buffer additives.

Amintra™ Affinity Resins
Expedeon’s offer range of Amintra resins (Ni-NTA, Cobalt, Glutathione, MBP and UFH).

Amintra™ Protein A Magnetic
Protein A is immobilized to highly porous and highly cross-linked agarose incorporating magnetic material and it can be used in any commercially available magnetic device.

Columns
Expedeon provides different columns, empty spin and gravity columns for use with any resin and desalting spin columns for sample preparation.

Protein Quantitation
Check our  BradfordUltra and BradfordMX Coomassie-binding colorimetric method for total protein quantitation.

Purification Kits
For conjugated antibodies, we offer AbSelect and AbPure™ purification Kits

For any questions regarding our products, please contact our technical support team at:

technical.enquiries@expedeon.com

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Products

View all products suitable for Affinity purification