Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target.
Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target (Figure bellow).
CIMac™ HDZ monoliths make HDZ-immobilized antibody columns even more effective. Because of their large channel size and the efficiency of convective mass transport, they eliminate the long loading residence times that are required for affinity chromatography on porous particle columns. Flow rates of 5–10 column volumes per minute allow complete purifications in a few minutes, even when the source material contains a low concentration of antigen. The same performance is achieved whether a small peptide or a large bio-assemblage like a virus particle or extracellular vesicle is isolated. The combination of HDZ monoliths and the immobilization protocol offers a strong tool for fast antigen isolation from complex biological sample (plasma, lysate, etc.) and consequently sensitive antigen quantification. An example of CIMac™ HDZ application is a purification of fibrinogen from human plasma.
There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ analytical chromatographic monolith.
Immunoaffinity columns using antibodies as ligands against mammalian proteins could be used for different applications in protein expression control and, if a standard available, for direct protein quantification in complex sample solutions. Additionally, these columns are ideal for polishing step of recombinant proteins, such as mammalian receptor Fc fusion proteins. Most importantly, such columns could extract a significant amount of a single membrane protein from native source, suitable for downstream analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against RAE-1 GPI anchored glycoprotein were developed (CIMmic HDZ-@RAE-1 column) as a part of Glycomet project with the main goal to analyze the antigen glycoprofile.
Bottom-up proteomic approach based on tandem mass spectrometry (MS/MS) is a method of choice for identification and quantitation of proteins in the complex biological samples. Followed by quantitative analysis of a protein sample, this approach enables the identification of putative biomarkers for early pathology diagnostics and differentiation. The main challenges confronting this analysis are suppression of low-abundance ions and the limited dynamic range of MS/MS.
Affinity depletion of abundant proteins is an important stage in routine sample preparation prior to tandem mass spectrometry (MS/MS) analysis of biological samples. One such protein is Human Serum Albumin (HSA). In this study, polyclonal αHSA antibodies were immobilised onto a chromatography support to use as immunoaffinity-based column for HSA depletion from embryo culture media for in vitro fertilization (IVF).
Yes. Make sure that either the inlet or the outlet of the column is open to prevent excess pressure buildup. Autoclaving monolithic columns for aseptic use is recommended. Prior to disposal, monoliths can also be autoclaved. For specifics, please consult firstname.lastname@example.org.