Products Used for Protein Quantitation

Protein purification is vital for the characterization of the function, structure, and interactions of proteins. The various steps in the purification process may include cell lysis, separating the soluble protein components from cell debris, and finally separating the protein of interest from product- and process-related impurities. Separation of the protein of interest, in its desired form, from all impurities, is typically the most challenging aspect of protein purification.

Order CaptureSelect® Affinity Products

Affinity chromatography is an effective technique for protein purification that often enables a single-step purification of proteins to a purity level sufficient for analytical characterization. Affinity chromatography is a separation technique based on molecular conformation—molecules that “fit” one another bind selectively in a “lock and key” fashion (e.g., an antibody may recognize and specifically bind an antigen). The technique can use application-specific chromatography resins that have antibody ligands (fragments of antibodies that contain the antigen-binding domain) attached to the resin surface. Most frequently, these ligands function with the target protein in a manner similar to that of antibody-antigen interactions. This highly specific fit between the ligand and its target compound enables affinity column chromatography that is also highly specific. Antigens bind to the resin-bound antibody ligand, while other sample components and impurities do not bind and flow through the affinity column. Bound antigen (typically the protein of interest) can then be eluted, often with a pH change that breaks the molecular antigen–antibody interaction, yielding a single, highly pure elution peak.

Life Technologies offers a variety of products for affinity chromatography through its CaptureSelect® product portfolio. CaptureSelect® products are affinity ligands based on camelid-derived (Figure 1) single-domain antibody fragments (VHH), created using proprietary technology. The affinity ligands are a 12 kDa single domain fragment that comprises the 3 complementarity-determining regions (CDRs) that form the antigen binding domain. These affinity ligands are efficiently produced in the yeast Saccharomyces cerevisiae and thus are not animal-derived. The affinity ligands are then covalently bound to chromatography beads, generating resins suited for protein purification via column chromatography.


Figure 1. Comparison of overall structure and major domains of human IgG and camelid Ig.

CaptureSelect® products possess a combination of unique properties, including selectivity, affinity, and stability, that provide competitive benefits: single-step purification, ease of use, minimized cost of purification, effective impurity removal, higher-quality product, and increased flexibility in the purification process.

These features make CaptureSelect® products the right tools for small-scale protein purification. The products are specific for a wide variety of target proteins and are therefore well suited to a variety of applications:

  • Small-scale protein purification—ready-to-use affinity resins, including:
  • Antibody Toolbox® products for the separation of immunoglobulin formats from a wide range of environments, such as plasma, milk from transgenic animals, or supernatant from mammalian cell cultures
  • Proteomics Toolbox® products for protein depletion to support biomarker research applications
  • HPLC analytical chromatography columns—rapid, sensitive, and precise quantitation and small-scale sample preparation of proteins from complex mixtures
  • Custom ligand design—the development of antibody ligands against a specific protein or impurity, enabling affinity purification tailored to novel applications

Most biomolecules can be purified with one of these products. Unlike the common bacterial coat protein components proteins A, G, and L, CaptureSelect® affinity ligands are developed with defined specificity and high avidity to the target, enabling the creation of affinity chromatography resins well suited for single-step protein purification (Tables 1 and 2).

  POROS® CaptureSelect® analytical columns Antibody Toolbox® Products Protein purification products Proteomics Toolbox® Products Conjugated ligands

Antibody analytics

  • Quantitation
  • PTM analysis


    

Antibody purification

  • Small scale
 
   

Protein purification

  • Natural/recombinant
  • Affinity tag
  


  
Biomarker discovery     
Instrumentation neededHPLC/FPLCFPLCFPLCFPLC Spin columnsELISA western SPR, BLI


BLI: bio-layer interferometry; PTM: post-translational modifications; SPR: surface plasmon resonance.

 

Table 1. Comparison of CaptureSelect® affinity products.
POROS® protein A, G resins POROS® CaptureSelect® ligands
Target type Prot A Prot G IgG Fc Kappa Lambda IgG-CH1 IgM
IgG1, IgG2, IgG4      
IgG3      
IgG, CH2 domain
deleted
       
IgM       
IgA, IgD, IgE      
Fab, F(ab´)2      
IgG Fc fusion protein     
Free light chains     

 

Table 2. POROS® CaptureSelect® analytical columns.