Vacuum Blotting Research Articles

Nonradioactive Northern Blot Analysis to Detect Ebola Virus Minigenomic mRNA.

In this chapter, we describe the detection of Ebola virus minigenomic mRNA using a nonradioactive Northern hybridization. This protocol comprises all steps beginning with the synthesis of a digoxigenin-labeled riboprobe, harvest of transcribed mRNA from cells transfected with the Ebola virus minigenome system, separation of mRNA species by denaturing RNA gel electrophoresis, transfer of the mRNA to nylon membranes by vacuum blotting, and finally the detection of minigenome-specific mRNA through hybridization with a labeled riboprobe directed against the reporter gene.This method allows the direct study of cis-acting regulatory regions as well as trans-acting factors involved in Ebola virus minigenome transcription compared to the indirect measurement of reporter protein activity that additionally reflects translational effects (see Chapter 6 in this book for details).

Molecular Mechanism of Signal Perception and Integration by the Innate Immune Sensor Retinoic Acid-inducible Gene-I (RIG-I)

RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5'-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA (dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5'-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products.

Assay of mucins in human tear fluid

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion ( r 2 > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Nonsense Suppression in Yeast Cells Overproducing Sup35 (eRF3) Is Caused by Its Non-heritable Amyloids

The [PSI+] prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils. Prion state of the Rnq1 protein, [PIN+], is required for the [PSI+] de novo generation but not propagation. Yeast [psi-] [PIN+] cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable [PSI+]. Here, we show that in such cells, most of Sup35 represents amyloid polymers, although the remaining Sup35 monomer is sufficient for normal translation termination. The presence of these polymers strictly depends on [PIN+], suggesting that their maintenance relies on efficient generation de novo rather than inheritance. Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization. About 10% of cells overproducing Sup35 form colonies on medium selective for suppression, which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants, allowing selection of cells deficient for soluble Sup35. A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced. The described polymers of these proteins differ from the [PSI+] polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.

Demonstration of Discrete Secreted and Membrane-Bound Ocular Mucins in the Dog

Our aims were to separate and characterize secreted canine ocular mucins, and to provide definitive evidence of membrane-bound mucins at the canine ocular surface. Mucus was collected by suction from the ocular surface of normal dogs and dispersed in guanidine hydrochloride and a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation separated secreted mucins from membranes, which were collected from the top of the gradients. Membranes were extracted with octyl glucoside and screened using lectins and anti-mucin antibodies. Gradient fractions containing secreted mucins were constituted into pools on the basis of differential lectin and antibody staining. High molecular weight material from each pool was purified by gel filtration. This material, and the membrane extract, were reduced and alkylated. Vacuum blotting of separated materials after agarose gel electrophoresis was used to compare subunit structure. Density gradient profiles indicated three principal secreted glycoprotein peaks: one staining strongly with anti-mucin antibodies. Gel filtration demonstrated that each contained high molecular weight material. Vacuum blots demonstrated the presence of two secreted glycoproteins with differently sized subunits. On the basis of buoyant density, one of these may be lipid complexed. Membrane extracted material stained with anti-mucin antibodies, and vacuum blotting of this material provided evidence for two membrane-bound components. In conclusion, we have shown that normal canine ocular mucus contains two secreted mucins, each exhibiting different subunit structure; one of these mucins may undergo lipid complexation. Normal canine ocular mucus also contains two membrane-bound mucins: one of which is unique among membrane mucins in showing subunit structure.

Effect of K+ depletion on glutamate dehydrogenase.

Slices of renal cortices were homogenized in Trisolv (Bioteck Laboratories, Houston,Tex., USA) at an approximate ratio of 1 g tissue/10 ml. RNA was precipitated with isopro-panol and washed twice with 75% ethanol. The electrophoresis of RNA was performed in1% agarose-formaldehyde gel, with 20 lg of RNA sample in each lane. After the electrophor-esis, RNA was transferred to a nylon membrane (maximum strength Nytran; Schleicher &Schuell, Keene, N.H., USA) with vacuum blotting. The membrane was prehybridized for2 h with 4¶standard saline citrate (SSC), 5¶Denhart’s solution, 40% formamide, 2.5% SDS,1¶blocking reagent (Bioteck Laboratories), 5% dextran, 0.05 mg/ml sheared salmon sperm,0.1 mg/ml Poly C, 0.05 mg/ml Torula yeast RNA and 20 m

A simplified method of analysis of cell-conditioned medium forInsulin-like Growth Factor-I (IGF-I) activity

To facilitate further investigation of the relationship between IGF-I and IGF binding proteins (IGFBPs) and their effect on myogenic satellite cells, we developed a procedure for rapid screening of cell-conditioned medium for free IGF-I. This method involves the adaptation and implementation of a DNA vacuum blotting manifold, which allows detection of a broad range of IGF-I and is rather sensitive (detection < 10 ng) when compared to western immunoblots. Additionally, this dot-blot method requires less than 40 minutes prior to the addition of the blotto-antibody mixture. Application of the dot-blot method for analysis of free IGF-I levels in conditioned medium samples offers an alternative to the use of western immunoblots, and may be used in initial screening of large numbers of conditioned medium samples.

Expression of the ob (obese) gene during lactation in mice.

The mutant gene responsible for the development of obesity in the ob/ob mouse has recently been localised and sequenced [l]. The identification of the ob (obese) gene provides an opportunity to make rapid progress in unravelling the critical factors involved in the regulation of body weight. The ob gene appears to be expressed exclusively in adipose tissue [ 1-31. where it codes for an 18,000 A4, protein which is secreted from the adipocyte as a 16,000 Mr product [I], termed leptin [4]. Leptin may act as a satiety factor [1,4-61, signalling the size of the white adipose tissue depots, but there is also evidence that it stimulates energy expenditure [4,5]. Increased expression of the ob gene has been observed following the administration of glucocorticoids [7], while fasting leads to a marked fall in gene expression which is reversed on subsequent re-feeding [3]. Cold exposure induces a substantial increase in energy expenditure and substrate flux in an animal, and we have recently found that expression of the ob gene is rapidly suppressed in the cold, a response which appears to be mediated by the sympathetic nervous system [8]. In the present study we have examined the effects of lactation, a physiological state which also induces major changes in nutrient flux and food intake in small animals [9], on the expression of the ob gene. Female mice of the 'Aston' variety were mated at 6-8 weeks of age. The main studies focused on mid-lactation (10-12 days postpartum), but analyses were also performed at early and late lactation (1-2 days and 18-20 days post-partum, respectively). The effects of abrupt weaning were examined in mid-lactation, tissue being taken 24 h after removal of the pups. Virgin mice, agematched to the lactating animals, were used as controls. The mice were killed by cervical dislocation and gonadal fat (and other tissues in pilot studies) rapidly removed and frozen in liquid N2. Total RNA was extracted and hctionated by agarose gel electrophoresis [3,8]. After vacuum blotting onto a charged nylon membrane (Boehringer Mannheim), the RNA was fixed with W light. The blots were probed with a 33-mer antisense oligonucleotide (5'-GGTCTGAGGCAGGGAGCAGCTCTTGGAGAAGGC) targeted to the mRNA encoding mouse ob mRNA [3,8]. The oligonucleotide was synthesised commercially, and labelled with DIG at the 5' end (R & D Systems Europe). Following post-hybridization washes, the membranes were processed essentially according to the protocols provided in the DIG detection kit (Boehringer), as described previously [3,8]. The membranes were immersed in 250 pM CDP-Stur (Tropix), a new chemiluminescence substrate. After incubation at 37°C for 15 min, the membranes were exposed to film at room temperature for up to 90 min, and bands on the film quantitated by densitometry. The blots were then stripped and sequentially reprobed for lipoprotein lipase mRNA and 18s rRNA using 30-mer and 31-mer antisense oligonucleotides, respectively [8]. The mRNA encoding ob was readily detected by chemiluminescence in white fat of female mice, but not in other tissues. using the 33-mer antisense oligonucleotide as a probe. ob mRNA was evident in gonadal adipose tissue throughout lactation. In three separate experiments the level of ob mRNA was

Analysis of Trinucleotide Repeats in Myotonic Dystrophy

Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat in the 3-untranslated region of a gene encoding a serine-threonine protein kinase. There is no one method to detect the entire range of expansion sizes possible in affected patients, so current diagnostic approaches rely on analyzing samples by hybridization of both polymerase chain reaction (PCR)-amplified CTG repeats (CTG-PCR) and genomic DNA. In this unit, the the Basic Protocol 1 describes the analysis of PCR-amplified repeats transferred to a nylon membrane by Southern blotting and hybridized to an alkaline phosphatase-labeled probe. The first support protocol describes a vacuum blotting technique for rapid transfer of the PCR product to the nylon membrane and the second support protocol describes the use of a radiolabeled oligonucleotide probe for hybridization. Analysis of genomic DNA by similar hybridization techniques is outlined in the second basic protocol. Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat Myotonic dystrophy is a genetic disorder characterized in 99% of clinically diagnosed families by an unstable CTG repeat.

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes

We describe a vacuum blotting procedure for transferring DNA fragments from conventional polyacrylamide sequencing gels to nylon membranes. The method employs a combination of vacuum-assisted diffusion (effected by a standard gel drier) and an osmotic gradient (effected by over- and underlying filters presoaked in ammonium acetate). Fragments up to 310 nucleotides in length transfer at 40–60% efficiency within 90 min. When combined with indirect end-labelling, the method allows genomic sequencing of a single-copy gene of Saccharomyces cerevisiae employing as little as 5 μg DNA per lane.

Vacuum blotting enhances nucleic acid transfer

Nucleic acids can be transferred from an agarose get to a transfer membrane using capillary Southern blotting or electroblotting techniques. Vacuum blotting represents an alternative method that offers a number of advantages.