New developments in Western blot technology

Western blotting is a highly valued method for protein identification and relative quantitation in complex samples. It combines size-based electrophoretic separation with immunoaffinity to identify specific proteins. This technique remains popular and has become a workhorse in biochemical research and clinical laboratories. Despite its utility and popularity, this method has many limitations including slow analysis, incompatibility with limited sample application, low throughput and low information content. Recently there has been significant success in improving different aspects of Western blotting. In this review, we provide an overview of the developments in the area of improving conventional Western blotting methods with a focus on recent developments in microfluidic Western blotting. We overview different separation platforms, and discuss studies on protein transfer methods as well as protein immobilization methods and chemistries. We also describe integrated miniaturized platforms that can perform rapid separations and immunodetections.     

Visible-Light Photoswitching by Azobenzazoles

Three visible-light responsive photoswitches are reported, azobis(1-methyl-benzimidazole) ( 1 ), azobis(benzoxazole) ( 2 ) and azobis(benzothiazole) ( 3 ). Photostationary distributions are obtained upon irradiation with visible light comprising approximately 80 % of the thermally unstable isomer, with thermal half-lives up to 8 min and are mostly invariant to solvent. On protonation, compound 1 H⁺ has absorption extending beyond 600 nm, allowing switching with yellow light, and a thermal half-life just under 5 minutes. The two isomers have significantly different pK_a values, offering potential as a pH switch. The absorption spectra of 2 and 3 are insensitive to acid, although changes in the thermal half-life of 3 indicate more basic intermediates that significantly influence the thermal barrier to isomerization. These findings are supported by high-level ab initio calculations, which validate that protonation occurs on the ring nitrogen and that the Z isomer is more basic in all cases.     

Boosting Conjugate Addition to Nitroolefins Using Lithium Tetraorganozincates: Synthetic Strategies and Structural Insights

We report the first transition metal catalyst- and ligand-free conjugate addition of lithium tetraorganozincates (R₄ZnLi₂) to nitroolefins. Displaying enhanced nucleophilicity combined with unique chemoselectivity and functional group tolerance, homoleptic aliphatic and aromatic R₄ZnLi₂ provide access to valuable nitroalkanes in up to 98 % yield under mild conditions (0 °C) and short reaction time (30 min). This is particularly remarkable when employing β-nitroacrylates and β-nitroenones, where despite the presence of other electrophilic groups, selective 1,4 addition to the C=C is preferred. Structural and spectroscopic studies confirmed the formation of tetraorganozincate species in solution, the nature of which has been a long debated issue, and allowed to unveil the key role played by donor additives on the aggregation and structure of these reagents. Thus, while chelating N,N,N’,N’-tetramethylethylenediamine (TMEDA) and (R,R)-N,N,N’,N’-tetramethyl-1,2-diaminocyclohexane (TMCDA) favour the formation of contacted-ion pair zincates, macrocyclic Lewis donor 12-crown-4 triggers an immediate disproportionation process of Et₄ZnLi₂ into equimolar amounts of solvent-separated Et₃ZnLi and EtLi.     

Synthesis,Structure, and DFT Analysis of the THF Solvate of 2-Picolyllithium: A 2-Picolyllithium Solvate with Significant Carbanionic Character

Previous studies of different solvates of 2-methylpyridyllithium (2-picolyllithium) have uncovered electronic structures corresponding to aza-allyl and enamido resonance forms of the metallated pyridine-based compounds. Here, we report the synthesis and characterization of [2-CH₂Li(THF)₂C₅H₄N], a new THF solvate. X-ray crystallographic studies reveal a dimeric arrangement featuring a non-planar eight-membered [NCCLi]₂ ring, in which the primary cation-anion interaction is between the central Li atom and the C atom of the deprotonated methyl group [length, 2.285(2) Å], suggesting a new carbanionic resonance structure for this 2-picolyllithium series. The significant carbanionic character of [2-CH₂Li(THF)₂C₅H₄N] was confirmed by gas-phase DFT calculations [B3LYP/6-311+G(d)] with the calculated electron density interrogated by means of quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses. For comparison these computational analyses were also performed on the literature structures of [2-CH₂Li(2-Picoline)C₅H₄N] and [2-CH₂Li(PMDETA)C₅H₄N]. In a reactivity study, [2-CH₂Li(THF)₂C₅H₄N] was found to undergo nucleophilic addition to pyridine to generate dipyridylmethane in a good yield.     

Lateral Metallation and Redistribution Reactions of Sodium Ferrates Containing Bulky 2,6-Diisopropyl-N-(trimethylsilyl)anilide Ligands

Alkali-metal ferrates containing amide groups have emerged as regioselective bases capable of promoting Fe−H exchanges of aromatic substrates. Advancing this area of heterobimetallic chemistry, a new series of sodium ferrates is introduced incorporating the bulky arylsilyl amido ligand N(SiMe₃)(Dipp) (Dipp=2,6-iPr₂-C₆H₃). Influenced by the large steric demands imposed by this amide, transamination of [NaFe(HMDS)₃] (HMDS=N(SiMe₃)₂) with an excess of HN(SiMe₃)(Dipp) led to the isolation of heteroleptic [Na(HMDS)₂Fe{N(SiMe₃)Dipp}]_∞ ( 1 ) resulting from the exchange of just one HMDS group. An alternative co-complexation approach, combining the homometallic metal amides [NaN(SiMe₃)Dipp] and [Fe{N(SiMe₃)Dipp}₂] induces lateral metallation of one Me arm from the SiMe₃ group in the iron amide furnishing tetrameric [NaFe{N(SiCH₂Me₂)Dipp}{N(SiMe₃)Dipp}]₄ ( 2 ). Reactivity studies support that this deprotonation is driven by the steric incompatibility of the single metal amides rather than the basic capability of the sodium reagent. Displaying synergistic reactivity, heteroleptic sodium ferrate 1 can selectively promote ferration of pentafluorobenzene using one of its HMDS arms to give heterotrileptic [Na{N(SiMe₃)Dipp}(HMDS)Fe(C₆F₅)]_∞ ( 4 ). Attempts to deprotonate less activated pyridine led to the isolation of NaHMDS and heteroleptic Fe(II) amide [(py)Fe{N(SiMe₃)Dipp}(HMDS)] ( 5 ), resulting from an alternative redistribution process which is favoured by the Lewis donor ability of this substrate.     

Adding a Structural Context to the Deprotometalation and Trans‐Metal Trapping Chemistry of Phenyl‐Substituted Benzotriazole

Organometallic bases are becoming increasingly complex, because mixing components can lead to bases superior to single‐component bases. To better understand this superiority, it is useful to study metalated intermediate structures prior to quenching. This study is on 1‐phenyl‐1H‐benzotriazole, which was previously deprotonated by an in situ ZnCl₂ ? TMEDA/LiTMP (TMEDA=N,N,N′,N′‐tetramethylethylenediamine; TMP=2,2,6,6‐tetramethylpiperidide) mixture and then iodinated. Herein, reaction with LiTMP exposes the deficiency of the single‐component base as the crystalline product obtained was [{4‐R‐1‐(2‐lithiophenyl)‐1H‐benzotriazole ? 3THF}₂], [R=2‐C₆H₄(Ph)NLi], in which ring opening of benzotriazole and N₂ extrusion had occurred. Supporting lithiation by adding iBu₂Al(TMP) induces trans‐metal trapping, in which C?Li bonds transform into C?Al bonds to stabilise the metalated intermediate. X‐ray diffraction studies revealed homodimeric [(4‐R′‐1‐phenyl‐1H‐benzotriazole)₂], [R′=(iBu)₂Al(μ‐TMP)Li], and its heterodimeric isomer [(4‐R′‐1‐phenyl‐1H‐benzotriazole){2‐R′‐1‐phenyl‐1H‐benzotriazole}], whose structure and slow conformational dynamics were probed by solution NMR spectroscopy.     

A machine for static and dynamic triaxial testing

A machine has been developed for studying the static and dynamic triaxial constitutive behavior of large specimens of geologic and construction materials. Test specimens can also contain a cylindrical tunnel cavity to permit study of tunnel-reinforcement structures and rock-structure interaction. The specimens are 0.3 m in diameter and 0.3 to 0.45 m high; the model tunnels can be up to 50 mm in diameter. Static and dynamic triaxial loads can be applied with maximum pressures of 200 MPa in static tests and 100 MPa in dynamic tests. Dynamic loading can also be superimposed on a static preload as large as 20 MPa. To facilitate study of tunnel reinforcement, the tunnel is maintained at ambient pressure, with access at both ends for instrumentation and photography. Example results show the influence on tunnel deformation of loading rate as well as the presence of joints and their orientation. For a given allowable tunnel closure, substantially greater pressures can be sustained under dynamic loading than under static loading, and substantially greater pressures can be sustained by an intact specimen than by a jointed specimen.     

The Effects of a Mathematics Infusion Curriculum on Middle School Student Mathematics Achievement

Increasing mathematical competencies of American students has been a focus for educators, researchers, and policy makers alike. One purported approach to increase student learning is through connecting mathematics and science curricula. Yet there is a lack of research examining the impact of making these connections. The Mathematics Infusion into Science Project, funded by the National Science Foundation, developed a middle school mathematics‐infused science curriculum. Twenty teachers utilized this curriculum with over 1,200 students. The current research evaluated the effects of this curriculum on students' mathematics learning and compared effects to students who did not receive the curriculum. Students who were taught the infusion curriculum showed a significant increase in mathematical content scores when compared with the control students.