NC Membranes Research Articles

Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10–25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Microstructural effects on permeability of Nitrocellulose membranes for biomedical applications

Nitrocellulose membrane (NC), as a paper-like matrix with microscale porous structures, has found widespread applications in biomedical fields due to its excellent biological features and physiochemical properties. In these biomedical applications, diffusion, convection, and binding reaction of biomolecules (e.g., acids and proteins) in NC through wicking flow is the fundamental physical process. However, the optimization of NC based biomedical devices has been limited by the lack of the understanding on the wicking flow behavior of NC membranes from the microstructural point of view. To address this, we experimentally and theoretically investigated the microstructural effects on the wicking flow behaviors (e.g., permeability, effective pore radius) of NC membrane and found that the wicking flow is highly dependent on the pore-morphology characterizing parameters (e.g., porosity and pore size). We further developed a theoretical model yielding a closed-form solution to predict the microstructure-permeability relation, which was validated by our experimental results. Our theoretical model would be a powerful tool for tailoring the wicking flow behavior of NC membranes through controlling the microstructural parameters, and thus for optimizing NC membrane-based biomedical devices from the view point of material design in the future.

A net fishing enrichment strategy for colorimetric detection of E. coli O157:H7

The strict regulatory requirements for pathogen monitoring in food systems to ensure safety demands that the detection method can recognize small numbers of pathogens. Although significant efforts on the development of biosensors have been reported with marked improvement in sensitivity, appropriate enrichment strategies are still needed in a detection scheme. Herein we proposed a unique strategy for the enrichment of pathogens, based on a concept termed as net fishing, to capture target bacteria with antibodies functionalized on porous substrates to fish the target from samples. Because of their large surface area porous nitrocellulose membranes (NC membranes) were used as substrates for target capture. By immersing the antibody modified NC membrane into liquid samples and upon mixing, we expect to anchor target pathogens on the membrane due to the specific interaction between antibody and target, where the nonspecific enrichment could be reduced compared to methods such as filtration. Meanwhile the net fishing enrichment requires 2h, less than common culture methods. An enzyme enhanced procedure was performed with the NC membrane after the capture to obtain colorimetric signal. Gold nanoparticle (GNP) probes conjugated with horseradish peroxidase (HRP) and antibody were used to label capture target bacteria, and the presence of pathogens was determined by the colorimetric signal generated from HRP catalyzed TMB reaction. With E. coli O157:H7 as target, we investigated the detection performance of the proposed net fishing strategy with an enzyme amplification step for colorimetric reporting. After optimization of detection conditions, we show that as low as 100CFU/ml E. coli O157:H7 could be detected. The feasibility was then demonstrated using pineapple juice as a food model.

A method to label biological molecules with dsDNA coated gold nanoparticles.

We described a new method to label biological molecules using gold nanoparticles (GNPs) and double stranded DNA. Researchers can conveniently label their own samples with GNPs using this method. The label is based on dsDNA with a 93.5% coverage of GNPs (dsDNA:GNP = 303:1). Antigens, streptavidin and biotin were labeled on GNPs and the success of the method was investigated with agarose gel electrophoresis, laser particle size analysis and ultraviolet spectrophotometry. These analyses confirmed that biological molecules were successfully bound to the GNPs. These molecules retained their biological activity and were able to detect targets on PVDF and NC membranes with excellent selectivity and low levels of background. Modified GNPs were also able to detect targets on nylon membranes, but with some degree of false positives. The maximum limit of detection was 25 ng proteins.

Controlling swelling behavior of poly (vinyl) alcohol via networked cellulose and its application as a reverse osmosis membrane

The hydrophilic nature of polyvinyl alcohol (PVA) makes it a promising membrane material for water treatment applications but its use is restricted by its high swelling capacity in water. We found that the swelling behavior of PVA can be controlled via aqueous blending of PVA with a modified form of cellulose-networked cellulose (NC). Dense PVA–NC membranes were obtained by varying NC concentration at 3, 10, 15, 20 and 30wt.%. Fabricated membranes were characterized for their mechanical, thermal and structural properties. It was found that 15wt.% of NC was optimum. The swelling capacity of PVA was reduced from 340% to 150%. The tensile strength of wet PVA improved 1520% and the elastic modulus improved 1400%. The fabricated membranes were tested for desalination by reverse osmosis (RO) using aqueous solution of sodium chloride with 25,000ppm feed concentration and an applied pressure of 24bars. A 272μm thick membrane allowed a water flux value of 1.2l/h·m2 with a salt rejection of 98.8%.

Electrophoretic interactions between nitrocellulose membranes and proteins: Biointerface analysis and protein adhesion properties

Protein adsorption onto membrane surfaces is important in fields related to separation science and biomedical research. This study explored the molecular interactions between protein, bovine serum albumin (BSA), and nitrocellulose films (NC) using electrokinetic phenomena and the effects of these interactions on the streaming potential measurements for different membrane pore morphologies and pH conditions. The data were used to calculate the streaming ratios of membranes-to-proteins and to compare these values to the electrostatic or hydrophobic attachment of the protein molecules onto the NC membranes. The results showed that different pH and membrane pore morphologies contributes to different protein adsorption mechanisms. The protein adsorption was significantly reduced under conditions where the membrane and protein have like-charges due to electrostatic repulsion. At the isoelectric point (IEP) of the protein, the repulsion between the BSA and the NC membrane was at the lowest; thus, the BSA could be easily attached onto the membrane/solution interface. In this case, the protein was considered to be in a compact layer without intermolecular protein repulsions.

Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter‐alpha‐trypsin inhibitor and saposin B but increased expression of an inter‐alpha‐trypsin inhibitor heavy chain 4 fragment

The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required.

Colorimetric sensor strips for lead (II) assay utilizing nanogold probes immobilized polyamide-6/nitrocellulose nano-fibers/nets

A facile, ultrasensitive, reproductive and selective sensor strip utilizing electrospun polyamide-6/nitrocellulose (PA-6/NC) nano-fibers/nets (NFN) membranes assemble bovine serum albumin decorated Au nanoparticles (BAu probe) for naked-eye colorimetric assay of Pb2+ has successfully prepared through dual-component alternate distribution multifluidic electrospinning technique. Benefiting from the extremely large specific surface areas and high porosity of NFN membranes, the stability of BAu probe dramatically increased and the strips presented a significant absorbance decreasing band at 546nm which induce the visual color changes from deep pink to white after incubated in Pb2+ liquor with a low detection limit of 0.2μM without any assistance of equipment. Upon exposure to a series of metal ions, only Pb2+ could induce a pink-to-white color change, which clearly exhibited that BAu probe immobilized PA-6/NC membranes could act as highly selective strips to detect Pb2+ with little interference from other metal ions. Additionally, the colorimetric responses are represented in visualized quantitative by calculated color difference from L⁎a⁎b⁎ coordinates which are presented with lightness and chrome values. Furthermore, the sensitivity of NFN membrane-based strips is much higher than that of film-based ones. The results indicate that this promising cost-effective sensing system could potentially allow for assaying of Pb2+ in human urine or blood as preliminary screening of lead poisoning.

Phagosomes Induced by Cytokines Function as anti-Listeria Vaccines

Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.

Labelling of antibodies and detection by laser ablation inductively coupled plasma mass spectrometry : PART III. Optimization of antibody labelling for application in a Western blot procedure

The labelling of three different polyclonal antibodies (chicken anti-bovine serum albumin, rabbit anti-chicken lysozyme and rabbit anti-bovine casein) with iodine and lanthanides has been investigated and the reaction conditions were optimized for application in a Western blot assay. For this purpose target protein standards (bovine serum albumin, chicken lysozyme and bovine β-casein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto NC membranes. After the immuno-reaction of the labelled antibodies with the antigen the element label is detected by laser ablation ICP-MS directly on the Western blot membrane. In this study it turned out that only the labelling of antibodies using p-SCN-Bn-DOTA (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) lanthanide chelates could be applied successfully in the Western blot approach whereas iodine labelling of the antibody showed unspecific binding and a too low sensitivity. For validation a conventional method, chemiluminescence detection, was applied and similar limits of detection at ng levels of antigen were achieved by both methods. In comparison to chemiluminescence detection, the Western blotting procedure in combination with LA-ICP-MS detection is less time consuming and the elemental signatures on the blots show long term stability. Last but not least LA-ICP-MS offers multiplexing capabilities and therefore simultaneous detection of three differentially labelled antibodies in one single Western blot assay is demonstrated.

Investigating the risk of two model virus in the laboratory

To study the survival time of recombination rival in environment and inactivation ability of different disinfectant and ultraviolet radiation against virus. NC membranes absorbed the recombinant adenovirus (rADV) or herpes simplex virus (rHSV) with green fluorescence protein (GFP) were laid, or immersed in various concentration of different disinfectants such as ethanol, sodium hypochlorite, lysol and geramine and then taked out them every 15 min, or exposed under ultraviolet radiation, then the NC membranes were adsorbed 1 h in cell, 37 degrees C 5% CO2 48 h. The results were observed under the fluorescence microscope. (1) the average survival time of rHSV under environment is less than 60 min, rADV is almost up to 2 h. (2) The infection ability of rHSV and rADV was inactived 15 min by both ethanol (100%, 70% and 50%) and sodium hypochlorite (5%, 2.5% and 1.25%). (3) Two virus can be killed by 0.1% bromogeramine. (4) Both 5% and 2.5% lysol, but rADV can not lost the infection on Vero Cell until 75 min by 1.25% Lysol. (5) The rHSV was inactivated under ultraviolet radiation, but rADV was not. The survival time of is different from both envelope rival and the no-envelope viral under nature environment and the inactivate ability of disinfectant also is different between two model virus; Disinfectant should be choose according to virus type.

Labelling of proteins by use of iodination and detection by ICP-MS

The labelling of three different proteins, bovine serum albumin (BSA), chicken egg white lysozyme and porcine gastric mucosa pepsin, with iodine using a commercially available reaction kit has been investigated for total protein amounts of 500 μg. The assay described here has already often been applied but with radioactive iodine isotopes and is extended in this work for the use of the stable isotope 127I and detection by ICP-MS. The reaction conditions are investigated and optimised for proteins separated by SDS-PAGE and electroblotted onto NC membranes. Detection has been performed by laser ablation ICP-MS on the membranes. Long reaction times result in degradation of proteins, whereas for short reaction times (4 min) integrated sensitivities of even 108 cps pmol−1 of protein have been realized. Total amounts of proteins have been investigated in the range from 0.015 (BSA) to 105 pmol (lysozyme). A calibration was performed for BSA from 0.015 to 15 pmol but the limit of detection was reached already at about 150 fmol due to high iodine blank values, only. The labelling procedure described here does not affect protein mobility in SDS-PAGE, because the change of the molecular weight is very moderate for the proteins investigated here. This is quite opposite to those procedures where chelating compounds are applied. ESI-FTICR-MS has been used to identify histidine and tyrosine as the target amino acids for iodination. Additionally, oxidation of methionine residues has been observed.

Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS

The labelling of two different proteins (bovine serum albumin, hen egg white lysozyme) with the commercially available chelating compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) has been investigated. The assay described here has been optimised for the application to detect proteins labelled by stable isotopes of Eu, Tb and Ho and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detection has been performed by laser ablation ICP-MS after electroblotting of the target proteins onto NC membranes. A range of total protein amounts from 0.015 pmol (BSA) to 105 pmol (lysozyme) has been covered. A calibration was performed for BSA in the range from 0.015 to 15 pmol and a limit of detection below 15 fmol can be estimated. For lysozyme integrated sensitivities of more than 107 cps pmol−1 of protein have been realized. The conditions, once optimised for labelling with Eu, have been applied for other lanthanides (Tb, Ho), too. ESI-MS of the intact and the tryptic digested lysozyme has been used as to provide a better understanding of the reaction chemistry and efficiency. The procedure described here looks promising to develop multielement labelling strategies (multiplexing) for ICP-MS applications in quantitative proteomics.

Swelling and mechanical behavior of modified poly(vinyl alcohol)/laponite nanocomposite membranes

AbstractNanocomposite (NC) membranes based on hydrophobically modified PVA and laponite were synthesized with varying laponite content in the feed. The incorporation of laponite in NC membranes was investigated by FTIR spectroscopy and thermogravimetric analysis. The swelling ratio of membranes was determined as a function of temperature and laponite content. Swelling studies of NC membranes exhibited the decrease in swelling with an increase in laponite content in the NC membranes. The swelling ratio of NC membrane with 20% laponite slightly increased with an increase in temperature. Dynamic mechanical analysis showed the systematic increase in storage modulus with laponite content, which indicates the enhancement of mechanical property upon laponite addition. There was also a decrease in the tan δ peak values of NC membranes with an increase in laponite content in NCs. The permeabilities through NC membranes as a function of solute size and laponite content were studied and the results showed molecular screening based on size. The permeability of solute reduced due to the presence of well‐dispersed laponite in the NC. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2896–2903, 2007

The 102 kDa Chicken Liver Acid Phosphatase: Dimeric Structure, Glycoprotein Nature and Immunological Properties

In this study we isolated a highly purified, homogeneous high molecular weight (HMW) AcPase (Mr 102 kDa) from the chicken liver. This enzyme was shown to be a slightly acidic (pI 5.0–6.1), dimeric sialoglycoenzyme, composed of two equivalent subunits. Its sugar moiety, characterized by interactions with specific lectins, was shown to be composed of hybrid and complex type of carbohydrate chains. Heterogeneity of the high molecular weight AcPase arising from variations of the sugar components was demonstrated by isoelectric focusing, followed by reactions of the isoelectric components with specific lectins on NC membranes. Structural relationship based on immunological similarities was shown between the HMW AcPases from carp, frog, and chicken livers.

Electroimmunoblotting of neuropeptide Y: Application to the rat vas deferens

In this study we have optimized the electroimmunoblotting conditions for neuropeptide Y (NPY). NPY standards and samples extracted from the rat vas deferens were separated on urea-sodium dodecyl sulphate gels. Densitometric scanning of the Coomassie Blue-stained gels allow a semi-quantitative analysis of NPY in the range of approximately 10(-11) to 10(-8) mol of NPY. Electroimmunoblotting of NPY was also shown to be best achieved overnight at 4 degrees C and with NC membranes of 0.22 micron. Under these conditions NPY extracted from the vas deferens has been efficiently electroimmunoblotted. Higher molecular weight NPY-reactive peptides were also detected that may be related to proteolytic processing of the NPY precursor.