转移缓冲液对蛋白质印迹的影响

利用三种转移缓冲液,分别以半干和湿转法,检测癌基因产物Bcl-2, 并获得不同强度的印迹结果.不含SDS的转移缓冲液转移效果明显优于含SDS的转移缓冲液,且SDS含量越高印迹带越弱;此外,半干转移与湿转相比,可大大缩短转移时间,但转移效果不及湿转理想且稳定性较差.     

Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

Intracellular Ca²⁺ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca²⁺ and Ca²⁺ buffers, with spatially discrete clusters of stochastic IP₃ receptor channels (IP₃Rs) controlling the release of Ca²⁺ from the endoplasmic reticulum. IP₃Rs are activated by a small rise of the cytosolic Ca²⁺ concentration and inhibited by large concentrations. Buffering of cytosolic Ca²⁺ shapes global Ca²⁺ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca²⁺ release, but affect the time course of the signals from the fluorescent Ca²⁺ indicator mainly by competing for Ca²⁺. At low [IP₃], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

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.     

Two-step purification of Cyanophora ferredoxin and its identification in soluble protein preparations by isoelectric focusing.

Cyanophora paradoxa ferredoxin is encoded by (cyano-)plastidic DNA, in contrast to those of all other photosynthetic eukaryotes investigated so far. In the present study we report (i) the rapid purification of a chloroplast-type [2Fe-2S] ferredoxin in a two-step procedure by DEAE-Sephadex and Mono Q ion-exchange chromatography; (ii) the biochemical characterization of the purified ferredoxin by electrophoretic separation methods on a microscale; and (iii) a qualitative and quantitative ferredoxin detection method in the femtomole range that allows densitometry, semidry immunoblotting, identification of ferredoxin in soluble cell protein preparations, and analysis of protein biosynthesis from cyanoplast poly(A)- RNA in vivo and in vitro. These fast micromethods should be useful for screening phototrophic species containing ferredoxins encoded by nonnuclear DNA.     

High-throughput immunoturbidimetric assays for in-process determination of polyclonal antibody concentration and functionality in crude samples

We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody 'concentration' and 'functionality' in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery. Possible influences from commonly employed buffers and salts (present in samples at various concentrations), and of pH variations, were studied for both assays. Interference effects were shown to be negligible for the 'concentration' assay, such that sample pre-treatment prior to assay is unnecessary. The 'functionality' assay displayed concentration dependent sensitivity to interference for ammonium sulphate and Tris-(hydroxymethyl)-amino-methane, but was essentially unaffected by all other salts and buffer combinations tested. The immunoturbidimetric assays described here are generically applicable to polyclonal antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation.     

Buffers and oscillations in intracellular Ca2+ dynamics

I model the behavior of intracellular Ca(2+) release with high buffer concentrations. The model uses a spatially discrete array of channel clusters. The channel subunit dynamics is a stochastic representation of the DeYoung-Keizer model. The calculations show that the concentration profile of fast buffer around an open channel is more localized than that of slow buffers. Slow buffers allow for release of larger amounts of Ca(2+) from the endoplasmic reticulum and hence bind more Ca(2+) than fast buffers with the same dissociation constant and concentration. I find oscillation-like behavior for high slow buffer concentration and low Ca(2+) content of the endoplasmic reticulum. High concentration of slow buffer leads to oscillation-like behavior by repetitive wave nucleation for high Ca(2+) content of the endoplasmic reticulum. Localization of Ca(2+) release by slow buffer, as used in experiments, can be reproduced by the modeling approach.     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II.

The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9. The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9. However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations. All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions. These results are interpreted in terms of different pathways for a rate-limiting proton transfer. In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64. Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants. The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants. The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups. Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient. At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64. The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively.     

An Efficient Low Cost Method for Gene Transfer to T Lymphocytes

Gene transfer to T lymphocytes has historically relied on retro and lentivirus, but recently transposon-based gene transfer is rising as a simpler and straight forward approach to achieve stable transgene expression. Transfer of expression cassettes to T lymphocytes remains challenging, being based mainly on commercial kits.

Aims

We herein report a convenient and affordable method based on in house made buffers, generic cuvettes and utilization of the widely available Lonza nucleofector II device to promote efficient gene transfer to T lymphocytes.

Results

This approach renders high transgene expression levels in primary human T lymphocytes (mean 45%, 41–59%), the hard to transfect murine T cells (mean 38%, 36–42% for C57/BL6 strain) and human Jurkat T cell line. Cell viability levels after electroporation allowed further manipulations such as in vitro expansion and Chimeric Antigen Receptor (CAR) mediated gain of function for target cell lysis.

Conclusions

We describe here an efficient general protocol for electroporation based modification of T lymphocytes. By opening access to this protocol, we expect that efficient gene transfer to T lymphocytes, for transient or stable expression, may be achieved by an increased number of laboratories at lower and affordable costs.     

The local Ca concentration profile in the vicinity of a Ca channel

Intracellular Ca signaling is governed by diffusion and buffering of Ca ions. Mobile endogenous buffers increase the redistribution of Ca ions and tend to restrict the regions of elevated Ca concentrations to the close vicinity of the channel. These Ca microdomains dominate fast Ca signaling, as observed, e.g., in synaptic transmission. The steady-state solution of the linearized differential equations of buffered Ca diffusion, as developed by Neher and Naraghi and Neher (1–3), will be reviewed and generalized to the case of more than two buffers. Immobile buffers do not enter the steady-state equations, but instead slow down Ca diffusion and prolong the time to reach the steady state. Based on this phenomenon, a quite different putative mechanism to localize Ca will be suggested that is likely to be realized in photoreceptors where Ca source, Ca sink, and Ca sensor form a complex, as was recently reported (4–6).     

A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose

An improved procedure for the electrophoretic transfer of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose is described. The use of more alkaline transfer buffers and the omission of an equilibration step before the transfer allow for the almost complete transfer of strongly basic proteins from gels to nitrocellulose without lowering the transfer efficiency for other proteins.     

Buffer solutes as stabilizers of 35S-amino acids: a study of volatility, radiochemical purity and biological activity

It is well established that volatile radioactive compounds are associated with preparations of 35S-amino acids, but misconceptions still exist. The addition of stabilizers to these preparations has created a false sense of security regarding the evolution of these volatiles from cell culture medium. Experiments presented here show that the commercially used buffers L-lysine, tricine and 3,4-pyridine-dicarboxylic acid all reduced, but did not eliminate, the evolution of 35S volatiles from tissue culture medium. In each case, the rate of release was approximately 12 nCi/mCi/day (or 0.001%), compared to approximately 44 nCi/mCi/day in the buffer's absence. None of six alternative buffers tested are more effective than the commercially used buffers. We found that the release of labeled volatiles is dependent on the nature of the culture medium, but is no greater from crude 35S-amino acid preparations than from purified 35S-methionine. These commercially used buffers are also effective in maintaining radiochemical purity and are compatible with biological activity; however, because these buffers do not entirely eliminate the release of radioactivity into the gas phase, routine safe-handling procedures should be practiced when working with these products.     

Ultrasonic absorption studies of protein-buffer interactions

The acoustic absorption of protein solutions in the presence of phosphate and other buffering ions has been studied in the physiological pH range. Buffers containing hydroxyl residues as titratable groups cause a pronounced increase of protein sound absorption, which is attributed to relaxation processes of proton transfer reactions between buffer ions and accessible imidazole and -amino groups of the protein surface. Amino group based buffers like Good's buffers do not induce additional sound absorption. Measurement of the ultrasonic absorption as a function of pH and of buffer concentration, and corresponding parameter fitting of the equation describing proton transfer relaxation processes has been used to evaluate equilibrium parameters. For the imidazole group of the amino acid histidine a pK value of 6.22 and for the imidazole group of the protein lysozyme a pK value of 5.71 have been determined. In hemoglobin the ligand-linked pK changes have been monitored by recording ultrasonic titration curves.     

The greater negative charge density of DNA in tris-borate buffers does not enhance DNA condensation by multivalent cations

As indicated by recent measurements of the electrophoretic free solution mobility, DNA appears to have a greater helical charge density in Tris-borate-EDTA (TBE) buffers than in Tris-acetate-EDTA (TAE) buffers. Since electrostatic forces play a major role in DNA packaging processes, we have investigated the condensation of closed circular plasmid DNA using total intensity and dynamic light scattering in Tris-borate, Tris-acetate, and Tris-cacodylate buffers with cobaltic hexa-amine (III) [Co(NH(3))(3+)(6)]. We find that neither the critical concentration of Co(NH(3))(3+)(6) nor the hydrodynamic radii of the resulting condensates vary significantly in the buffer systems studied here despite the prediction that DNA condensation should occur at significantly lower Co(NH(3))(3+)(6) concentrations in Tris-borate buffers. Assuming a persistence length behavior similar to B-DNA in the presence of multivalent cations, a decrease in the attractive counterion correlation pressure decay length in Tris-borate buffers does not account for our observations. It is possible that the binding of multivalent cations to DNA may hinder borate association with the DNA double helix.     

An optimized strategy for ICAT quantification of membrane proteins

The work presented here focuses on the development of a method adapting isotope labeling of proteins with ICAT to the study of highly hydrophobic proteins. Conditions for the labeling of proteins were first established using two standard soluble proteins and iodoacetamidyl-3,6-dioxaoctanediamine biotin (PEO-iodoacetyl biotin). Results demonstrated the efficiency of the labeling in the presence of high concentrations of both SDS and urea. These conditions were then used to label a highly hydrophobic mitochondrial membrane protein, the adenine nucleotide translocator ANT-1, with PEO-iodoacetyl biotin and then with the cleavable ICAT reagent. The results presented here show that labeling of proteins with cleavable ICAT is possible and may even be improved in strong denaturing buffers containing both SDS at a concentration higher than 0.5% (w/v) and urea. These results open the possibility of applying the ICAT strategy to complex samples containing very hydrophobic proteins solubilized in urea-SDS buffers. The adaptability of the developed method is demonstrated here with preliminary results obtained during the study of membrane-enriched fractions prepared from murine embryonic stem cells.     

The Impact of the Invasive Alien Plant,Impatiens glandulifera,on Pollen Transfer Networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward.     

Micro quantitative Edman manual sequencing

A manual Edman technique is described which allows sequential quantitative determination of from 3 to 10 amino terminal residues on quantities of peptides or proteins down to one nanomole. This is achieved by a fast, efficient method of obtaining the anilinothiazolinone or phenylthiohydantoin amino acid, and quantitating by either back hydrolysis and amino acid analysis or by a new, rapid, high resolution, quasi-isocratic, high-pressure liquid chromatographic procedure. The overall method has been extensively tested successfully on both peptides and proteins of known and unknown amino-terminal sequence and the results included here. In addition, a wide variety of applications relevant to primary structure analysis such as sequencing blocked polypeptides, use of denaturing agents as coupling buffers, reduction of protein or peptide losses on consecutive sequencing and peptide mixture analysis are all incorporated in the methodology outlined.     

Control of enzyme ionization state in supercritical ethane by sodium/proton solid-state acid–base buffers

We have previously demonstrated that a solid-state buffer could be successfully used to control the ionization state of subtilisin Carlsberg cross-linked microcrystals (CLECs) suspended in supercritical ethane (sc-ethane) in the presence of acid–base active species such as salt hydrates and zeolite molecular sieves. Here we studied the effect of six zwitterionic proton/sodium (pH–pNa) solid-state acid–base buffers on the catalytic activity of subtilisin CLECs in sc-ethane at high and low water activity (a_W). CLECs were strongly activated by increasing a_W. At high a_W, and despite the high hydrolysis rates, transesterification activities were still about one order of magnitude higher than those observed at lower a_W. This is in contradiction with what was previously reported in the absence of acid–base control and supports the hypothesis that the poor catalytic performance of subtilisin CLECs at high a_W observed in those studies was due to the inhibitory effect of the hydrolytic by-product, rather than to the competition of water with propanol for the acyl-enzyme intermediate. Although the catalytic activity of subtilisin showed a general positive correlation with the aqueous pK_a of the acid–base buffers tested here, our results also show that as expected, the acid–base behavior of the buffers in nonaqueous media is more complex than what can be predicted from aqueous-based parameters alone. This work further confirms the usefulness of solid-state acid–base buffers in supercritical biocatalysis but highlights the need for further research on the topic.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.