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Associated Methods
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
A number of different methods for eluting antibodies from sections have been tried. These include acid and alkaline buffers, chaotropic agents, urea, and guanidin hydrochloride, alone or in various combinations. A common finding has been that these methods may work well with low-avidity antibodies, but that they all are inefficient in removing high-avidity antibodies. The situation is somewhat analogous to affinity purification of antibodies (Chapter 4, Section V. A) and illustrates well the strength of interaction between highly avid antibodies and their antigens. In fact, as discussed in the preceding chapter, graded urea treatment is useful for removing some unspecific staining but does not affect specific staining much if avid antibodies are used.
Peptide Separation by Reverse-Phase High-Performance Liquid Chromatography
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Phosphate or phosphoric acid has been found to give particularly good results in HPLC of peptides.14,23-26 It is presumed that ion-pairing between peptide groups and the hydrophilic anion increase the polarity of the migrating species, thus decreasing their retention. Some workers add perchlorate as a chaotropic agent to phosphate mobile phases.27-29 Trifluoroacetic acid also works well;21 larger perfluoridated acids have been used to provide more hydrophobic counterions and thus increase retention of hydrophilic peptides.18,30,31
RNA
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
It is noteworthy that the MS2 RNA was purified efficiently by chromatography on benzoylated-naphthoylated DEAE cellulose (Sedat et al. 1967). Later, Hung (1969) elaborated a simple DEAE-cellulose method for isolating biologically active RNA from the phages Qβ and MS2. A chromatographic procedure at different temperatures with columns of cellulose CF-11 was able to distinguish the extent of the RNA secondary structure as tested with the phage f2 RNA, among other RNA models (Engelhardt 1972). The adsorption chromatography of nucleic acids, and the Qβ RNA among them, on siliconized porous glass was elaborated as a useful method for the separation of tRNA, rRNA, and mRNA (Mizutani 1983). The MS2 RNA was employed as a model by the separation of single- and double-stranded RNA forms, which was based on their differential binding to silica particles at high concentration of the chaotropic agent guanidinium thiocyanate (Beld et al. 1996). The presence of divalent Ca2+ cations in solutions greatly enhanced the deposition kinetics of the MS2 RNA on silica surfaces (Shen et al. 2011). Furthermore, the MS2 RNA was employed by the elaboration of the nucleic acid separations using superficially porous silica particles (Close et al. 2016).
Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency
Published in mAbs, 2019
Chen Chen, Tetsuya Wakabayashi, Masaru Muraoka, Feng Shu, Chia Wei Shan, Chong Chor Kun, Ching Tim Jang, Ishin Soehano, Yuichiro Shimizu, Tomoyuki Igawa, Jun-Ichi Nezu
We hypothesized that when the electrostatic and/or hydrophobic interactions of proteins are controlled, the proteins can be separated according to the valency of the binding between Protein L resin and the κ LC. However, we also considered that chaotropic agents may have a negative impact on the physico-chemical properties of proteins depending on its strength and concentration, so we prioritized controlling the conductivity during acid elution using mildly chaotropic salt based on Hofmeister series, such as sodium chloride (NaCl), when testing this hypothesis.22–24 Furthermore, for the same reason, we kept the conductivity level below the physiological level. We also noted reports showing that, when IgG was purified with Protein A, elution efficiency was highly compromised when a high concentration of salt was added to the acid because the hydrophobic interaction was overly enhanced, which was another reason to keep the salt concentration at less than the physiological level.25
Inter-assay variability in automated serum free light chain assays and their use in the clinical laboratory
Published in Critical Reviews in Clinical Laboratory Sciences, 2020
Laura Caponi, Nadia Romiti, Elona Koni, Annarita Di Fiore, Aldo Paolicchi, Maria Franzini
The possibility of converting all FLC dimers to monomers using detergents or reductants that break the disulfide bonds, thus overcoming the different sensitivities of the assays against them, is unlikely to be feasible. Mild detergents are already used in current assays to reduce the background (e.g. in the N-Latex assay), while strong detergents, chaotropic agents and thiol reductant (which would also reduce intra-chain disulfide bonds) would denature conformational epitopes and would make the analyte immunologically different with respect to the antigen used for immunization. In addition, thiol reductant would break the disulfide bonds of the antibodies, thus releasing FLC from the immunoglobulins present in serum, which are far more abundant than FLC.
Unraveling the complexity of the extracellular vesicle landscape with advanced proteomics
Published in Expert Review of Proteomics, 2022
Julia Morales-Sanfrutos, Javier Munoz
The most popular approach for digesting EVs-enriched preparations is in-solution digestion in MS-compatible buffers using urea [51], rapigest [52] and even trifluoroethanol [17]. Figure 2 Alternatively, FASP has been also widely adopted for the analysis of EVs [53]. Unfortunately, detergents and chaotropic agents are often incompatible with MS and require removal. This inevitably increases handling and subsequent peptide/protein loss, might introduce biases and are time-consuming. These issues have fostered the development of novel methodologies including the integrated Stage-Tip (iST) protocol [54] and the single-pot solid-phase-enhanced sample preparation (SP3) [55]. These approaches are simple and minimize handling while maintaining a high sample recovery and therefore are well suited for the analysis of EVs preparations [56,57]. As discussed above, one of the main potential of EVs is in the field of biomarkers. However, the robustness, reproducibility, sensitivity, and especially, high-throughput needed to analyze large cohorts of samples with minute amounts have sometimes preclude large-scale clinical studies. Recently, technological developments in robotic liquid handling stations have provided an end-to-end automated method that is ideal for these studies. These platforms can exploit the benefits of paramagnetic SP3 beads [58] and deliver peptides ready for MS analysis in 96-well plate format with minimal human intervention. Therefore, it is probably a matter of time that these robotic systems will be adopted and introduced in biomarker proteomic studies of EV-related particles.(2) Mass spectrometry