Ceruloplasmin
René Lontie in Copper Proteins and Copper Enzymes, 1984
The first purifications of Cp were achieved before chromatographic procedures were available and depended on precipitation steps.3,22 Later it was found that the protein was one of the most acidic in serum and binds to the top of an anion exchanger as a blue band if whole serum is applied. Most methods for Cp preparation depend on the use of columns of diethylaminoethyl cellulose or Sephadex®.23-27 Broman28 introduced hydroxyapatite chromatography in the preparation scheme when he discovered that it separated Cp into a major and a minor component (Section II.F.2). Apatite chromatography has, however, not been of general use in purifications, which implies that a large number of investigations have been carried out on a mixture of the two forms. Cation-exchange chromatography provides a final step to remove additional impurities.27,29 An alternative is gel filtration where aggregates and apoprotein elute slightly ahead of the main peak. In Uppsala we have employed a modified version30 of the method published by Broman and Kjellin,31 since it gives large amounts of pure protein in a comparatively short time (Scheme 1).
Separation Of The Bound And Unbound Forms Of The Radioactivity
Erwin Regoeczi in Iodine-Labeled Plasma Proteins, 2019
Many proteins possess an affinity for a specific adsorbent, a property that can come in handy for getting rid of the unreacted radioactivity. Sometimes a simple cation-exchange chromatography (e.g., on sulfopropyl-Sephadex®) will do, as for example in the case of certain hormones,34 and a-thrombin.35,36 Affinity chromatography can be carried out in other instances with the help of small but strong columns. Thus plasminogen can be captured on Sepharose®-lysine and eluted subsequently by e-aminocaproic acid;37 antithrombin III can be adsorbed on Sepharose®-heparin and regained by salt elution.38 These are just a few examples of the temporary immobilization technique that can be applied to a number of proteins, provided the medium is inert toward the nonprotein radioactivity and the protein- bound radioactivity is fully retrievable without harsh treatment. (In this writer’s experience, insulin, for example, is not fully recoverable after adsorption on to talc.39) Adsorbing out the protein, instead of the nonprotein radioactivity, from the reaction mixture can be of advantage with regard to quality control; by applying special elution techniques (e.g., a series of steps), it is often possible to find out at an early stage whether the labeling has modified the binding properties of the protein.
Using iodine for analysis
Tatsuo Kaiho in Iodine Made Simple, 2017
Ion chromatography is a type of liquid chromatography which can qualify and quantify anions such as chloride ions, fluoride ions, and sulfate ions and cations such as sodium ions and ammonia ions with high sensitivity. Ion chromatography uses ion-exchange resin as the stationary phase. By utilizing the difference in the time each substance remains on the ion-exchange resin according to the strength of the charge, substances within a sample can be separated. In the suppressor between the column and the detector, ions which were originally included in the mobile phase are removed and placed on the detector.
In silico prediction of post-translational modifications in therapeutic antibodies
Published in mAbs, 2022
Developability assessments aim to identify candidates with long-term stability, manufacturability, and low heterogeneity.9 Forced degradation with thermal, pH, and light stress has been used to accelerate chemical degradation and identify liable residues.10 Peptide mapping can identify the specific sites for chemical modifications after forced degradation. In contrast, chromatographic techniques such as cation exchange chromatography and hydrophobic interaction chromatography can monitor the overall change in charge and hydrophilic variants, respectively.11 However, experimental approaches for identifying PTM liabilities are time-consuming and require high quantities of the purified protein.12 The sample preparation and data analysis for peptide mapping is incredibly labor-intensive.13 At earlier stages of drug development, the number of forced degradation conditions is limited by the low availability of the purified protein.10 Computational tools are becoming more common during developability assessments due to the low cost, lack of sample consumption, and high speed. In the past decade, computational tools have been used to predict PTM liable sites and engineer antibodies with better chemical stability.14
Current challenges in biotherapeutic particles manufacturing
Published in Expert Opinion on Biological Therapy, 2020
Mafalda G. Moleirinho, Ricardo J.S. Silva, Paula M. Alves, Manuel J. T. Carrondo, Cristina Peixoto
Ion-exchange chromatography (IEC) is one of the most widely used chromatographic techniques for purification of large particles which exploits the differences in particle’s charge [97]. Anion or cation exchange chromatography techniques can be applied depending on the net charge of the particle to be purified. The net charge at neutral pH will depend on the particle surface composition which differs from each viral particle and serotype. Most of the viral particles have an isoelectric point below 7.4, being negatively charged at physiological pH. Adenovirus, AAV, retrovirus, lentivirus and baculovirus are some examples of viral particles that have been successfully purified using anion exchange chromatography [35].
HbA1c method performance: The great success story of global standardization
Published in Critical Reviews in Clinical Laboratory Sciences, 2018
Emma English, Erna Lenters-Westra
Methods based on charge differences depend on the extra negative charge that occurs when glucose is attached to the N-terminal valine of the HbA β-chain. Examples of such methods are cation-exchange chromatography and electrophoresis. Cation-exchange chromatography is a process that allows the separation of a mixture of proteins based on the charge properties of the molecules in the mixture. Charged Hb and other Hb components are eluted at varying times depending upon the net charge of the molecule in relation to a gradient of increasing ionic strength buffers passed through a cation-exchange column.
Related Knowledge Centers
- Chromatography
- Elution
- Inorganic Chemistry
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- Nucleotide
- Protein
- Amino Acid
- Ion
- Chemical Polarity
- Moiety