Home About us Contact
|
Home About us Contact | ||
|
|
||
Electrophoresis Separation (electrophoresis + separation)
Selected AbstractsPump-free and low-cost negative pressure sampling device for rapid sample loading in MCEELECTROPHORESIS, Issue 24 2009Hongmei Hu Abstract A pump-free and low-cost negative pressure sampling device for injecting well-defined non-biased sample plugs into the separation channel of MCE was developed. It was composed of a pipet bulb, a 3-way electromagnetic valve and a single voltage supply at constant voltage. A sub-atmospheric pressure was created by hand-pressing air out of the pipet bulb and retained in it by switching the 3-way electromagnetic valve at cutoff position. During the sample loading stage, the sub-atmospheric pressure in the pipet bulb was applied via a 3-way electromagnetic valve to the headspace of the sealed sample waste reservoir (SW). A pinched sample plug was formed at the channel intersection in less than 0.5,s. Once the 3-way electromagnetic valve was switched to connect SW to ambient atmosphere to release the vacuum in SW, electrophoresis separation was consequently activated under the electric potentials applied. Experimental results demonstrated the pump-free negative pressure sampling device worked well in a wide vacuum degree ranged from ,250 to ,30,mbar with a satisfactory analytical precision. The sample consumption for each cycle was calculated to be 51,12,nL under the sampling pressure. Theoretical deduction indicates that the volume of the pipet bulb can be further reduced to 1,mL, which is critical for minimizing the sampling device for MCE. [source] Application of dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium in wall modification for capillary electrophoresis separation of proteinsELECTROPHORESIS, Issue 3 2005Wei Wei Abstract A zwitterionic surfactant, dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium (C12H25N+(CH3)2CH2CHOHCH2SO3,), named dodecyl sulfobetaine (DSB), was used as a novel modifier to coat dynamically capillary walls for capillary electrophoresis separation of basic proteins. The DSB coating suppressed the electroosmotic flow (EOF) in the pH range of 3,12. At high DSB concentration, the EOF was suppressed by more than 8.8,times. The DSB coating also prevented successfully the adsorption of cationic proteins on the capillary wall. Anions, such as Cl,, Br,, I,, SO42,, CO32,, and ClO4,, could be used as running buffer modifiers to adjust the EOF for better separation of analytes. Using this dynamically coated capillary, a mixture of eight inorganic anions achieved complete separation within 4.2,min with the efficiencies from 24,000 to 1,310,000,plates/m. In the presence of ClO4, as EOF adjustor, the separation of a mixture containing four basic proteins (lysozyme, cytochrome c, ,-chymotrypsinogen,A, and myoglobin) yielded efficiencies of 204,000,896,000,plates/m and recoveries of 88%,98%. Migration time reproducibility of these proteins was less than 0.5% relative standard deviation (RSD) from run to run and less than 3.1% RSD from day to day, showing promising application of this novel modifier in protein separation. [source] Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresisELECTROPHORESIS, Issue 9 2004Leyi Gao Abstract A fast and simple method for the direct, simultaneous detection of nitrite (NO2,) and nitrate (NO3,) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO2, and NO3, in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm×75 ,m ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 ,M. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer. [source] A simple polyacrylamide gel electrophoresis procedure for separation of polyamidoamine dendrimersELECTROPHORESIS, Issue 16 2003Ajit Sharma Abstract A simple, inexpensive, and rapid electrophoresis technique was developed for use as a routine tool for evaluating purity of polyamidoamine (PAMAM) dendrimers. A variety of factors influencing migration of generations 0,7 dendrimers on nongradient polyacrylamide gels were evaluated. The low generation dendrimers were found to be very sensitive to diffusion during or after electrophoresis. The proposed method incorporates steps that minimize diffusion, in order to obtain improved resolution and sensitivity, especially for the lower-molecular-weight dendrimers. This was accomplished by inclusion of a dendrimer fixation step with glutaraldehyde and performing the electrophoresis separation, fixation, staining, and destaining at 4°C. PAMAM dendrimer separation was studied under basic and acidic conditions. Electrophoresis under acidic conditions gave increased resolution and sensitivity over separation at alkaline pH. Oligomers and trailing generations could be clearly separated and visualized under these conditions. The smallest PAMAM dendrimer, generation 0, was visible at 1.5 ,g under the optimized acidic conditions. With slight modifications, this technique should be applicable to separation of other water-soluble dendrimers. [source] Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2004Natacha Turck Abstract Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations. [source] mRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] Differential Capture of Serum Proteins for Expression Profiling and Biomarker Discovery in Pre- and Posttreatment Head and Neck Cancer Samples,THE LARYNGOSCOPE, Issue 1 2008Gary L. Freed MD Abstract Introduction: A long-term goal of our group is to develop proteomic-based approaches to the detection and use of protein biomarkers for improvement in diagnosis, prognosis, and tailoring of treatment for head and neck squamous cell cancer (HNSCC). We have previously demonstrated that protein expression profiling of serum can identify multiple protein biomarker events that can serve as molecular fingerprints for the assessment of HNSCC disease state and prognosis. Methods: An automated Bruker Daltonics (Billerica, MA) ClinProt matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer was used. Magnetic chemical affinity beads were used to differentially capture serum proteins prior to MALDI-TOF analysis. The resulting spectra were analyzed using postprocessing software and a pattern recognition genetic algorithm (ClinProt 2.0). An HNSCC cohort of 48 sera samples from 24 patients consisting of matched pretreatment and 6 to 12 month posttreatment samples was used for further analysis. Low-mass differentially expressed peptides were identified using MALDI-TOF/TOF. Results: In the working mass range of 1,000 to 10,000 m/z, approximately 200 peaks were resolved for ionic bead capture approaches. For spectra generated from weak cation bead capture, a k-nearest neighbor genetic algorithm was able to correctly classify 94% normal from pretreatment HNSCC samples, 80% of pretreatment from posttreatment samples, and 87% of normal from posttreatment samples. These peptides were then analyzed by MALDI-TOF/TOF mass spectometry for sequence identification directly from serum processed with the same magnetic bead chemistry or alternatively after gel electrophoresis separation of the captured proteins. We were able to compare this with similar studies using surface-enhanced laser desorption ionization (SELDI)-TOF to show this method as a valid tool for this process with some improvement in the identification of our groups. Conclusions: This initial study using new high-resolution MALDI-TOF mass spectrometry coupled with bead fractionation is suitable for automated protein profiling and has the capability to simultaneously identify potential biomarker proteins for HNSCC. In addition, we were able to show improvement with the MALDI-TOF in identifying groups with HNSCC when compared with our prior data using SELDI-TOF. Using this MALDI-TOF technology as a discovery platform, we anticipate generating biomarker panels for use in more accurate prediction of prognosis and treatment efficacies for HNSCC. [source] Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillaryELECTROPHORESIS, Issue 15 2006Mahmoud M. Yassine Abstract Preparative capillary zone electrophoresis separations of cytochrome,c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C14DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7,million plates/m were observed in 50-µm,id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1,g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome,c resulted in dramatic improvements in the efficiency and peak shape. Using 100,mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH,7.0 with a 100-µm,id capillary, the maximum sample loading capacity in a single run was 160,pmol (2.0,µg) of each protein. [source] | ||||||||||