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At least 19 recordsLinked to original sources
PubMed1991

SDS-Minigel electrophoresis of human tears. Effect of sample treatment on protein patterns.

An automated Minigel electrophoresis system (PhastSystem, Pharmacia, Uppsala, Sweden) was tested for human tear protein analysis. Tear samples were treated under nonreducing or reducing conditions before sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE). Micro-amounts of tears (2 microliters) were sufficient for analysis and separation and visualization of proteins were completed within 2 hr. Tear proteins were identified using purified control proteins and immunoblotting techniques, using antisera against immunoglobulin (Ig) A (alpha) heavy chains, Ig heavy and light chains, secretory components and lactoferrin. In nonreduced tears, lactoferrin (seen as a double band), serum albumin, tear-specific prealbumin (TSPA), and lysozyme were clearly separated. Secretory IgA (sIgA) was seen as a smear on top of the gel. Immunostaining also showed a major Ig light chain containing protein. After reduction, the protein profiles showed marked changes. In reduced tears, immunoglobulin heavy and light chains (molecular weight [MW]: 64 and 28 kD, respectively) were detected on the SDS-PAGE profile after immunostaining, and represented disulfide cleavage fragments, which originated from sIgA. Reduction resulted in the liberation of the secretory component piece (MW: 85 kD), which was found to co-migrate with the tear lactoferrin bands. Both lactoferrin and serum albumin acted as larger proteins on SDS-PAGE after reduction. The authors found that the two methods of sample treatment, before electrophoresis, resulted in marked differences on the electropherograms.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Modification of a discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide gel system for minigel electrophoresis.

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system was recently described by J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271). The system was developed to separate with high and broad resolution the components from large volume samples after an overnight electrophoresis. This system was found to have many advantages. However, when used directly as a minigel system, this method cannot sustain the high voltage inherent to minigel electrophoresis and produces artefacts, namely a double front and a loss of resolution in the low molecular weight range. These problems were eliminated using the buffer system of M. A. Porzio and A.M. Pearson [1977) Biochem. Biophys. Acta 490, 27-34) in the separating gel and in the electrode chambers. The resulting modified discontinuous minigel system has the same advantages as the technique described for large slab gel electrophoresis, including the effective and rapid transfer of high molecular weight proteins to nitrocellulose membranes, as well as the advantages of the minigel format.

Animals↗
PubMed2006

DNA fingerprinting of Vibrio cholerae and Aeromonas species by pulsed-field minigel electrophoresis.

DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.

Aeromonas↗
PubMed1986

An exponential gradient marker for use with minigel polyacrylamide electrophoresis systems.

Minigel electrophoresis has reduced the required sample size and cost of running polyacrylamide gels without loss of resolution. The many problems incurred in the minigel system when pouring multiple gradient gels are eliminated by pouring individual gels with the described economical, exponential gradient marker, which can accurately deal with the small gel volumes. The advantages of exponential gradients are discussed.

Buffers↗
PubMed2026

Attomolar Detection of HIV-1 With Label-Free RCA-rCRISPR on Smartphone.

HIV remains a major global public health challenge, causing 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions due to the lack of inexpensive and equipment-free detection methods. Here, we developed a low-cost, robust, and label-free CRISPR-based diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy combines rolling circle amplification (RCA) with plasmid reporter-based ratiometric CRISPR (rCRISPR) that enables the detection of HIV RNA down to single-digit aM sensitivity from PWH-derived HIV samples ex vivo. Unlike conventional RCA, which requires fragmentations of long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the long HIV genome without pre-fragmentation. Cas12a reaction then detected RCA products by converting supercoiled ΦX174 plasmid reporters to relaxed forms. The target concentration was quantified based on the supercoil-to-relaxed plasmid ratio. Further, we constructed an all-in-one smartphone-based minigel electrophoresis device to demonstrate equipment-free HIV viral load testing. Finally, the assay has demonstrated for BRAF point mutation detection, showcasing the robustness of our strategy for broad disease diagnostic applications.

CRISPR↗
PubMed2025

Attomolar Detection of HIV-1 with Label-Free RCA-rCRISPR on Smartphone.

Human Immunodeficiency Virus-1 (HIV) remains a major global public health challenge, having led to over 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions, disproportionately affecting vulnerable populations and underserved communities across the world. This illustrates the critical need for accessible, accurate, and equipment-free diagnostic tools to enhance detection and thus provide opportunities to curb its spread. Here, we developed a low-cost, robust, and label-free rolling circle amplification (RCA)-rCRISPR diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy, combining the integration of RNA-detecting RCA reaction with plasmid reporter-based ratiometric CRISPR (rCRISPR), enables sensitive detection of unprocessed RNA targets without the need for intensive sample pre-treatment. This label-free RCA-rCRISPR diagnostic platform detected HIV RNA down to single-digit aM sensitivity (~3000 copies/mL) from PWH-derived HIV samples ex vivo. Unlike typical RCA, which requires sample fragmentations to break long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the entire HIV genome without pre-fragmentation. For point-of-care (POC) applications, we constructed an all-in-one smartphone-based minigel electrophoresis device to facilitate equipment-free HIV viral load testing, making it accessible to resource-limited communities. Additionally, the assay has demonstrated the ability for point mutation detection (BRAF mutation in canine urothelial carcinoma), showcasing the robustness of our strategy for broad disease diagnostic applications.

HIV↗
PubMed2003

Kinetworks protein kinase multiblot analysis.

The proteomics analysis of protein kinases and other cell-signaling proteins in tumor samples by traditional two-dimensional (2-D) gel electrophoresis is complicated by the low abundance of these regulatory proteins relative to metabolic enzymes and structural proteins. We present an antibody-based method called Kinetworks that relies on sodium dodecyl sulfate (SDS)-poly-acrylamide minigel electrophoresis and multilane immunoblotters to permit the specific and quantitative detection of 45 or more protein kinases or other signal transduction proteins at once. The technique can also permit the resolution of these proteins based on differences in their phosphorylation state and other forms of covalent modification. Kinetworks profiling of protein kinases in solid human tumors and cell lines can reveal profound differences in their expression and phosphorylation states, which can serve for the identification of cancer diagnostic markers and therapeutic targets for drug discovery.

Biomarkers, Tumor↗
PubMed1993

Rapid classification of phenylketonuria genotypes by analysis of heteroduplexes generated by PCR-amplifiable synthetic DNA.

We describe a rapid and simple method for phenylketonuria genotyping which identifies five point mutations within exon 12 of the human phenylalanine hydroxylase gene. The method involves PCR amplification of the target exon and hybridization with a PCR-amplifiable synthetic DNA (universal heteroduplex generator, UHG). The UHG contains identifiers consisting of nucleotide substitutions and/or deletions, contiguous with known mutation sites within the target exon. DNA heteroduplexes are resolved by nondenaturing polyacrylamide minigel electrophoresis. Individual mutant genotypes are identified by characteristic banding patterns, in either homozygous or heterozygous states. The method may potentially be applied to rapid genotyping of any mutation or series of mutations within PCR-amplifiable genetic material.

Base Sequence↗
PubMed1993

HLA-DQA1 allele and suballele typing using noncoding sequence polymorphisms. Application to 4AOHW cell panel typing.

HLA-DQA1 typing of the 4AOHW cell panel is presented using a novel strategy that exploits both intron and exon polymorphisms. Intron sequences adjacent to the variable HLA-DQA1 second exon exhibit stable polymorphisms that are specific for locus alleles and certain suballelic DR/DQ haplotypes. A PCR-RFLP method has been developed that is based on amplification of a 780-bp segment extending from intron 1 through exon 2 to intron 2. Stable sequence polymorphisms provide restriction enzyme sites and confer mobility variations detected on polyacrylamide minigel electrophoresis. Direct band comparison of amplified products and restriction fragments with known standards facilitates pattern comparison, obviating the requirement for accurate molecular weight determination. This method, using only two enzymes, identifies a total of 11 allelic and suballelic groups, including all eight DQA1 alleles encoded at the second exon.

Alleles↗
PubMed1995

Rapid genotype analysis in type 2B von Willebrand's disease using a universal heteroduplex generator.

A new diagnostic technique based on DNA heteroduplex analysis has been used to identify specific point mutations in the von Willebrand's factor (vWF) gene of patients with von Willebrand's disease type 2B. Molecular analysis in these patients has shown previously that their mutations are clustered in a short region of sequence in exon 28 of the vWF gene. The principle of the method involves heteroduplex formation between amplified genomic sequence containing the defect and an exon 28 vWF gene universal heteroduplex generator (UHG). The UHG is a synthetic vWF gene exon 28 homologue which contains a number of sequence mismatches designed to generate allele specific heteroduplexes for each type 2B mutation. Individual mutant genotypes are identified by characteristic banding patterns following polyacrylamide minigel electrophoresis. The technique is rapid, simple, inexpensive, and is ideally suited for adoption by non-specialist haematology laboratories for screening purposes.

Base Sequence↗
PubMed1996

A rapid HLA-DRB1*04 subtyping method using PCR and DNA heteroduplex generators.

We describe here a rapid polymerase chain reaction (PCR)-based method for the identification of HLA-DRB1*0401-*0412 alleles. The method is based on the generation of specific DNA heteroduplex patterns between PCR products derived from selective group-specific amplification of the various DRB1*04 alleles and PCR products derived from two synthetic DNA heteroduplex generator (DHG) molecules following non-denaturing polyacrylamide minigel electrophoresis. One DHG was designed to detect DRB1*0401, *0405, *0407, *0408, and *0409 alleles, whilst the other was designed to detect DRB1*0402, *0403, *0404, *0406, *0410, *0411, and *0412 alleles. Characteristic heteroduplex patterns were obtained for all DRB1*04 alleles tested both in homozygous and heterozygous situations. Both DHG and PCR-SSP (sequence-specific primer) typing were performed on 41 DRB1*04 positive DNAs from Singaporean Chinese blood donors and complete concordance in results was obtained. HLA-DRB1*0403, *0405, and *0406 were found to account for 95% of the DRB1*04 alleles in the population studied. The DHG technique described is technically simple and rapid since it essentially involves only two PCR amplifications per individual subtyping. The technique is particularly useful for resolving DRB1*04 combinations which are indistinguishable by PCR-SSO (sequence-specific oligonucleotide) or PCR-SSP subtyping.

Alleles↗
PubMed1993

Electrophoretic separation of rat skeletal muscle myosin heavy-chain isoforms.

A new technique for the sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation of rat skeletal muscle myosin heavy-chain (MHC) isoforms is presented. This technique allows for the separation of the four identified MHC isoforms known to be present in adult rat skeletal muscle. These types of MHC are commonly called I, IIa, IIx or IId, and IIb. The procedure can be performed using minigel electrophoresis systems and does not involve preparation of gradient-separating gels or the use of special cooling devices. The procedure accommodates both silver and Coomasie Blue staining. Thus the procedure is simple to perform and highly repeatable, providing high-resolution separation of MHC protein isoforms. The percent composition of the four adult MHCs in rat soleus, medial gastrocnemius, diaphragm, and levator ani muscles by use of this procedure and Coomasie Blue staining is similar to that previously reported. This new technique provides a novel and easy-to-perform method for the separation of rat skeletal muscle MHC isoforms.

Animals↗
PubMed1991

Detection of Epstein-Barr virus sequences in primary brain lymphoma without immunodeficiency.

We searched for Epstein-Barr virus (EBV) sequences by enzymatic DNA amplification in nine primary brain lymphomas from patients without immunodeficiency. We used seven nonlymphoma brain tumors as negative controls, and the Raji cell line as a positive control. We detected EBV DNA, using ethidium bromide-stained-agarose minigel electrophoresis and dot blot hybridization, in the positive control and in only one brain lymphoma tumor; we did not detect EBV DNA in the other tumors. The EBV-positive patient had a second B-cell monoclonal population in the peripheral blood without detectable EBV DNA, suggesting a direct role for EBV in the development of the brain lymphoma.

Brain Neoplasms↗
PubMed1993

Separation and characteristics of glycoproteins in tears which inhibit coating and precipitation of protein.

Using a modified turbidimetric assay to determine the protein concentration in human tears by precipitation with trichloroacetic acid (TCA) we found lower protein concentrations if compared with other methods for protein determination. This implies that a factor in human tears is able to inhibit the precipitation of protein by TCA. Earlier a coating inhibitory factor in human tears was described which is able to prevent coating of a polyacrylate surface by proteins using a ELISA methodology. Because of the similarity in its behaviour towards protein we investigated whether the same factor could be responsible for both inhibitory effects. A pool of human tears was separated into various fractions using HPLC whereafter inhibitory activity in the turbidimetric and the coating assay could be found in the same fractions. Characterization of the inhibitory factor was performed by minigel-electrophoresis (SDS-PAGE), after which blotting and staining with a lectin (Jacalin) revealed two subunits of a glycoprotein with a molecular weight of 30 and 70 kD. The inhibitory factor also could be isolated if human tears were incubated for 30 min at 100 degrees C whereafter precipitated protein was removed by centrifugation. Inhibitory activity could be detected in the supernatant and an identical glycoprotein profile could be produced after staining with lectin (Jacalin). The results of this study suggest that a soluble glycoprotein serves as a coating and precipitation inhibitor in tears and may play an important role in the protein to protein interaction on the surface of the eye.

Chemical Precipitation↗
PubMed1990

Selective binding of a 30-kilodalton protein to disposable hydrophilic contact lenses.

To investigate the accumulation of tear proteins on disposable extended-wear contact lenses (42% Etafilcon A and 58% hydration), a technique involving sodium dodecyl sulfate-polyacrylamide minigel electrophoresis combined with a sensitive silver-staining method was used. Besides the binding of large amounts of tear lysozyme the authors found an accumulation of an as yet unidentified 30-kilodalton (kD) protein. Longitudinal experiments showed lysozyme binding after 1 day of lens wear. The 30-kD protein was detected after a 2-day wearing period. The fact that protein deposition occurs during the relatively short wearing period of these lenses (1 week) may explain the unexpectedly high incidence of contact lens-associated conjunctivitis observed with these lenses.

Contact Lenses, Extended-Wear↗
PubMed1997

A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed, paraffin-embedded tumors.

Evaluation of the clinical significance of p53 gene mutations requires molecular analysis of primary tumors from large series of patients with long follow-up periods. In this study, we describe a sensitive, rapid, nonisotopic and inexpensive procedure for the polymerase chain reaction (PCR)-single-stranded conformational polymorphism (SSCP) detection and subsequent sequencing of p53 mutations in formalin-fixed and paraffin-embedded tumor (PET) samples. To optimize this method, we used a panel of 34 mutations previously identified in this gene by isotopic PCR-SSCP analysis of frozen colorectal carcinomas. Identical SSCP band shifts were observed in PET sections from each of these frozen tumors, except for one case in which the tumor cell content was probably too low. All of the 34 mutations were detected by use of an optimized minigel SSCP/silver staining procedure. The total PCR-SSCP screening time with use of this protocol was less than 8 hours. An additional improvement was the use of multiplex SSCP to screen two p53 exons (5/7 and 6/8) simultaneously, thus effectively halving the amount of reagents and time required for mutation analysis. To identify the exact nucleotide alterations, mutant single-stranded DNA was excised from silver-stained SSCP gels, reamplified, and used as template in sequencing reactions. The PCR-SSCP procedure we describe can be performed in routine histopathology laboratories, requiring only a thermal cycler and minigel electrophoresis apparatus. Our results demonstrated the feasibility of conducting large retrospective analyses of p53 gene mutation status in archival tumor material. Studies of this kind should allow elucidation of the clinical role of p53 mutation in various human cancer types.

Carcinoma↗
PubMed1990

A vertical submarine electrophoresis apparatus for polyacrylamide minigels.

A vertical submarine electrophoresis apparatus for use with minislab polyacrylamide gels is described. The design allows polyacrylamide gels to be run with the same ease and convenience that agarose gels are run with horizontal submarine apparatuses. The vertical submarine features a single buffer chamber with a restriction between the upper and the lower portions of the chamber. Acrylamide gels, cast between 9 X 10-cm glass slides, are inserted into the restriction and are completely immersed in buffer. Thus, current flows primarily through the gel itself, but some current flows through the buffer in the restriction surrounding the gel. Because water-tight separation of buffer chambers is not necessary, time-consuming and/or expensive procedures such as sealing with agarose or using fragile notched glass plates are eliminated. The apparatus can be set up to run a gel in less than 30 s. It is versatile in that gels of varying thickness (0.5, 0.8, 1.5, and 3 mm) can be run on a single apparatus. The apparatus has been used for sodium dodecyl sulfate gels, low ionic strength native gels for nucleoprotein complexes, and composite acrylamide-agarose gels.

Chemistry Techniques, Analytical↗
PubMed2005

Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples.

The random intergenic spacer analysis is a recently developed technique for the study of microbial populations. The bacterial intergenic spacer (ITS) is located between 16S rRNA and 23S rRNA genes and presents different length and sequence among bacterial species. Therefore, the amplicons can be separated by electrophoresis commonly performed at low voltage during several hours. Although this technique is especially useful for unculturable microorganisms, it has not been applied before to clinical sample analysis. As these samples have a limited number of bacterial species, the size of the gels may be reduced to facilitate their handling and to reduce the running time. To obtain maximum separation among the ITS bands, we analysed in this work different electrophoretical conditions including staircase electrophoresis, a technique based on the application of several voltage steps. The results obtained showed a different behaviour of the electrical resistance during the performance of submarine horizontal and vertical staircase electrophoresis. In the first case the resistance decreased during most of the running time whereas in the second case it increased. Here, we show that the performance of horizontal staircase electrophoresis reduces the running time more than 80% with respect to conventional electrophoresis at low voltages. This procedure was applied to the separation of ITS bands from bacterial DNA present in a tissue from a vocal cord biopsy. The sequencing of these bands allowed their identification. This new procedure may be very useful in the rapid diagnosis of bacteria present in human, animal and plant tissues.

Bacteria↗