The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.
In isothermal non-coalescence behaviours of a droplet against a wall, an air film of micrometre thickness plays a crucial role. We experimentally study this phenomenon by letting a droplet levitate over a moving glass wall. The three-dimensional shape of the air film is measured using an interferometric method. The mean curvature distribution of the deformed free surface and the distributions of the lubrication pressure are derived from the experimental measurements. We vary experimental parameters, namely wall velocity, droplet diameter and viscosity of the droplets, over a wide range; for example, the droplet viscosity is varied over two orders of magnitude. For the same wall velocity, the air film of low-viscosity droplets shows little shape oscillation with constant film thickness (defined as the steady state), while that of highly viscous droplets shows a significant shape oscillation with varying film thickness (defined as the unsteady state). The droplet viscosity also affects the surface velocity of a droplet. Under our experimental conditions, where the air film shape can be assumed to be steady, we present experimental evidence showing that the lift force generated inside the air film balances with the droplet’s weight. We also verify that the lubrication pressure locally balances with the surface tension and hydrostatic pressures. This indicates that lubrication pressure and the shape of the free surface are mutually determined. Based on the local pressure balance, we discuss a process of determining the steady shape of an air film that has two areas of minimum thickness in the vicinity of the downstream rim.
Therapeutic antibodies are prone to degradation via a variety of pathways during each stage of the manufacturing process. Hence, a low-cost, rapid, and broadly applicable tool that is able to identify when and how antibodies degrade would be highly desirable to control the quality of therapeutic antibody products. With this goal in mind, we have developed signature-based sensing system to discriminate differently degraded therapeutic antibodies. The use of arrays consisting of conjugates between nanographene oxide and fluorophore-modified single-stranded DNAs under acidic pH conditions generated unique fluorescence signatures for each state of the antibodies. Multivariate analyses of the thus obtained signatures allowed identifying (i) common features of native, denatured, and visibly aggregated antibodies, (ii) complicated degradation pathways of therapeutic omalizumab upon time-course heat-treatment, and (iii) the individual compositions of differently degraded omalizumab mixtures. As the signature-based sensing has the potential to identify a broad range of degraded antibodies formed by different kinds of realistic stress types, this system may serve as the basis for high-throughput assays for the screening of antibody manufacturing processes.
Liquid droplets of protein–polyelectrolyte complexes (PPCs) have been developed as a new candidate for stabilization and concentration of protein drugs. However, it remains unclear whether additives affect the precipitation and redissolution yields of PPCs. In the present study, we investigated the PPC formation of human immunoglobulin G (IgG) and poly-L-glutamic acid (polyE) in the presence of various additives that have diverse effects, such as protein stabilization. Alcohols, including ethanol, successfully increased the PPC precipitation yield to over 90%, and the PPCs formed were completely redissolved at physiological ionic strength. However, poly(ethylene glycol), sugars, and amino acids did not improve the precipitation and redissolution yields of PPCs over those observed when no additives were included. Circular dichroism spectrometry showed that the secondary structure of polyE as well as electrostatic interactions play important roles in increasing the PPC precipitation yield when ethanol is used as an additive. The maximum concentration of IgG reached 100 mg/ml with the use of ethanol, which was 15% higher efficiency of the protein yield after precipitation and redissolution than that in the absence of additives. Thus, the addition of a small amount of ethanol is effective for the concentration and stabilization of precipitated PPCs containing IgG formulations.
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