Electrophoresis, a transport technology that transitioned from moving boundary method to zone method.

Gel electrophoresis, a transport technology, is one of the most widely used experimental methods in biochemical and pharmaceutical research and development. Transport technologies are used to determine hydrodynamic or electrophoretic properties of macromolecules. Gel electrophoresis is a zone technology, where a small volume of sample is applied to a large separation gel matrix. In contrast, a seldom-used electrophoresis technology is moving boundary electrophoresis, where the sample is present throughout the separation phase or gel matrix. While the zone method gives peaks of separating macromolecular solutes, the moving boundary method gives a boundary between solute-free and solute-containing phases. We will review electrophoresis as a transport technology of zone and moving boundary methods and describe its principles and applications.

Elucidating the mechanisms of additive effects at high concentrations on hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is a commonly used chromatography technique for purifying proteins. It utilizes salting-out salts to facilitate the binding of native proteins to weakly hydrophobic ligands. There have been three proposed mechanisms for the promoting effects of salting-out salts, which include the dehydration of proteins by salts, cavity theory, and salt exclusion. To evaluate the above three mechanisms, an HIC study was conducted on Phenyl Sepharose using four different additives. These additives included a salting-out salt (NH4)2SO4, sodium phosphate that increases the surface tension of water, a salting-in salt MgCl2, and an amphiphilic protein-precipitant polyethylene glycol (PEG). Results indicated that the first two salts resulted in protein binding, while MgCl2 and PEG led to flow-through. These findings were then used to interpret the three proposed mechanisms, which showed that MgCl2 and PEG deviated from the dehydration mechanism, and MgCl2 also deviated from the cavity theory. The observed effects of these additives on HIC were reasonably well explained for the first time by their interactions with proteins.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and ß-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Non-Affinity Purification of Antibodies.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis.

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle.

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis.

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.

Gel-electrophoresis based method for biomolecular interaction.

Electrophoresis is one of the most important analytical technologies for characterization of macromolecules and their interactions. Among them, native gel electrophoresis is used to analyze the macromolecules in the native structure. It differs in principle and information from those obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or blue native polyacrylamide gel electrophoresis (BN-PAGE). SDS-PAGE is carried out in the presence of strong denaturant, SDS, while BN-PAGE is done in the presence of negatively charged dye, e.g., Coomassie brilliant blue, G-250. Here, we describe native gel electrophoresis using agarose gel and a buffer at pH 6.1 composed of histidine and 2-(N-morpholino) ethanesulfonic acid. First, a protocol for vertical and horizontal formats of agarose native gel electrophoresis is described followed by different staining procedures. Then, various examples obtained using the developed procedure will be shown to demonstrate how the technology can be applied to specific cases and the advantages or caveats of the present technology.

Western blotting of native proteins from agarose gels.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis.

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein. One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

A rabbit monoclonal antibody-mediated lateral flow immunoassay for rapid detection of CTX-M extended-spectrum ß-lactamase-producing Enterobacterales.

Infections of CTX-M extended-spectrum ß-lactamase-producing Enterobacterales are a severe threat in clinical settings. CTX-M genes on plasmids have been transferred to many Enterobacterales species, and these species have spread, leading to the global problem of antimicrobial resistance. Here, we developed a lateral flow immunoassay (LFIA) based on an anti-CTX-M rabbit monoclonal antibody. This antibody detected CTX-M variants from the CTX-M-9, CTX-M-2, and CTX-M-1 groups expressed in clinical isolates. The LFIA showed 100% sensitivity and specificity with clinical isolates on agar plates, and its limit of detection was 0.8 ng/mL recombinant CTX-M-14. The rabbit monoclonal antibody did not cross-react with bacteria producing other class A ß-lactamases, including SHV. In conclusion, we developed a highly sensitive and specific LFIA capable of detecting CTX-M enzyme production in Enterobacterales. We anticipate that our LFIA will become a point-of-care test enabling rapid detection of CTX-M in hospital and community settings as well as a rapid environmental test.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis.

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

Western blotting analysis of proteins separated by agarose native gel electrophoresis.

Western blotting was attempted to analyze proteins separated by agarose native gel electrophoresis that was previously developed on His/Mes buffer system. This report shows a simple protocol for blotting agarose native gel to a PVDF membrane by soaking the gel in sodium dodecylsulfate-containing transfer buffer and 3 examples of such analysis. First example showed expression of a recombinant antibody in HEK293 cells by direct staining of the agarose native gels for both proteins and nucleic acids and staining of the blots for proteins and host cell proteins. These analyses demonstrated usefulness of agarose native gel electrophoresis, confirming that the recombinant antibody migrates toward the cathode while nucleic acids and a majority of host cell proteins migrate toward the anode. Second example demonstrated the phosphorylation state of MAP kinase in human lymphocyte cell line. Namely, agarose native gel can separate kinase, whose phosphorylation can be analyzed by Western blotting. Third example showed correlation of Escherichia coli ß-galactosidase expression between the oligomerization and enzyme activity using antibody and substrate staining.

Agarose native gel electrophoresis for characterization of antibodies.

This study was conducted to evaluate applicability of the previously reported native agarose gel electrophoresis to the analysis of various monoclonal and polyclonal antibodies. Experiments were carried to test the electrophoresis system for characterization of different monoclonal antibodies and animal serum, analysis of expressed antibodies in cell culture and evaluation of antibody stability. An attempt to optimize the electrophoretic condition was made by adjusting the electrode buffer concentration, electrophoretic run time and agarose concentration.

Improving the quality of a recombinant rabbit monoclonal antibody against PLXDC2 by optimizing transient expression conditions and purification method.

Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems.

Biophysical characterization of a model antibody drug conjugate.

Antibody drug conjugates (ADC) are important next-generation biopharmaceuticals and thus require stringent structure characterization as is the case for monoclonal antibodies. We have tested several biophysical techniques, i.e., circular dichroism, analytical ultracentrifugation, differential scanning calorimetry and fluorescence spectroscopy, to characterize a fluorescein-labeled monoclonal antibody as a model ADC. These techniques indicated possible small structure and stability changes by the conjugation, while largely retaining the tertiary structure of the antibody, consistent with unaltered biological activities. Thus, the above biophysical techniques are effective at detecting changes in the structural properties of ADC.

Capto MMC mixed-mode chromatography of murine and rabbit antibodies.

Murine antibodies have weak affinity for Protein-A. Here, we have tested binding of murine monoclonal antibody (mAb) to Protein-A or Protein-A/Protein-G mixture under salting-out conditions. The addition of ammonium sulfate to HEK conditioned medium (CM) expressing murine mAb resulted in complete binding, leading to its elution by low pH or neutral arginine solution. Alternatively, a mixed-mode chromatography using Capto MMC resin was developed as a capture step. Binding of murine mAb occurred at neutral pH. The bound mAb was eluted with a gradient from 0.3 M NaCl to 0.3 M arginine/0.3 M NaCl at pH 7.0. The Capto MMC-purified murine mAb was further purified by hydroxyl apatite chromatography. Similarly, rabbit mAb was processed with some modifications. Binding of rabbit mAb to Capto MMC required a lower pH. Elution of the bound rabbit mAb was achieved by a gradient to 0.3 M NaCl, pH 7.0.

Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.