Slot blotting

Hybridization can be performed by merely spotting the sample to a membrane, where it is immobilized by baking and subsequently hybridized to a suitable probe. Sample application can be performed with commercially available manifolds that apply sample into multiple wells of the manifold and let sample migrate as spots or slots into the membrane hence the name dot blot or slot blot hybridization (W2). The sample wells are repeatedly washed prior to removing the membrane to bake or irradiate in order to fix the sample, which is then ready for hybridization with probe. 

RT-PCR, Southern blot, slot blot, immunobiot, and ICC reveal correlation of mRNA and CYP2D6 protein across 13 brain regions higher expression is seen in brains from alcoholics versus nonalcoholics. In hippocampus this was localized in CAl-3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites (Miksys et al., 2002). 

Stains in the form of colloidal dispersions The need for highly sensitive detection methods for proteins after blotting (e.g., electroblotting, dot-blotting, slot blotting) applied to nitrocellulose or PYDF membranes drove scientists attention to develop new stains. It was discovered that the stains in the form of colloidal dispersions are suitable for such applications. The most commonly used colloidal stains are... 

A number of methods are available for analyzing tumor HER-2 status. The selection of the method depends on the target molecule to be detected. The target molecules are DNA mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot (Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al 1993), fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH) on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction (Gramlich et al., 1994). Assays to determine mRNA oveiexpiession include Northern blot (Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and ISH (Naber et al., 1990). Methods to assess HER-2/mcm protein product overexpression... 

Hybridization analysis using immobilized DNA includes dot blots and slot blots. Dot and slot blots are named for the circular and slotted well shapes in the templates used to present test samples to a membrane surface. They eliminate the need for restriction digestion and electrophoretic resolution steps by depositing samples of DNA to be tested directly onto a hybridizable membrane surface. Dot blot methodology is faster and easier for hybridization screening than Southern blotting, especially when many samples are to be screened... 

The assay of enzyme induction at the mRNA level is much easier to perform than the chemical assay of each individual enzyme and the response to a physiological event is intrinsically faster to detect. Moreover, mRNA is often more stable under cell extract preparation conditions than enzymes, if destruction by RNAses can be prevented. For analysing a number of different enzymes in parallel, only one sample preparation procedure is necessary, which produces a crude RNA extract, and only in the final step the different DNA or RNA probes are used for detection of specific enzyme RNAs. Recently, the detection of RNA is further facilitated by ready-to-use RNA isolation kits, non-radioactive DNA or RNA probes and the dot-blot and slot-blot techniques, respectively, [51,52]. 

Wash the blot twice, 5 min each with agitation, at 650 in wash buffer 1 (1 mAf EDTA, 5% SDS, 40 mAf NaHP04, pH 7.2). It is most convenient to heat the buffer to 65 °C in a microwave oven and do the wash in a plastic container, on a shaker, at room temperature. Use 125 ml wash buffer for each 1200 cm2 of membrane (one slot-blot sheet). 

Various formulations of nylon membrane filters have appeared on the market in recent years.32 The filter material is less brittle than nitrocellulose, and the stable linkages formed with DNA allow stripping off probes by heat while still retaining the target DNA on the filter, thus allowing reuse. The membranes have proved particularly effective for Southern blot hybridizations and are quite amenable to use in slot-blot and dot-blot devices, which allow application of up to 96 DNAs on a single filter. The membranes can be incubated in thermostable plastic pouches that require only a small volume of buffer. However, quantitation of DNA hybridizations on these filters has been no better than for nitrocellulose (C. P. Kurtzman, unpublished, 1987). 

RNAs are denatured by formaldehyde/heat treatment. All of the stock solutions for RNA slot blots are made using sterile diethyl pyrocarbonate (DEPC)-treated water. RNAs are diluted as necessary from stock solutions with water, and 3 volumes of 6.15 Mformaldehyde in 10X standard saline citrate (SSC) is added to give a final RNA concentration of 10-100pg/ml. The RNA dilutions are heated to 65° for 15 min and quick-chilled on ice. The denatured stock is further diluted with 4.16 M formaldehyde in 7.5 X SSC such that the desired concentration of RNA may be applied to each slot in a total volume of 400 pi. The nylon membrane is piewet in water and then soaked in 10X SSC for 20 min. Slot blots are performed using a commercially available apparatus hooked to a vacuum source. After the samples are blotted through, each well is washed with 400 pi of 10 X SSC. The membrane is removed from the apparatus and baked in a vacuum oven at 80° for 2 hr. 

The highly radioactive end-labeled oligonucleotides should not be used for more than 1 week after preparation because they are subject to radiochemical degradation. Labeled probes not used immediately are stored at — 20°. We routinely prepare several groups of slot blots and perform the hybridization to oligonucleotide probes on the day they are labeled. 

As an additional characterization, we hybridize the allele-specific probes to rDNA Southern blots to confirm that they hybridize to restriction fragments containing the region from which they were designed. The Southern blots are hybridized and washed using the same conditions described for RNA slot blots. 

An Immuno-Slot-Blot Assay for Detection and Quantitation of Alkyldeoxyguanosines in DNA... 

The immuno-slot-blot assay, a noncompetidve immunosorbent assay first described by Rajewsky and coworkers (3) and further developed in our laboratory (4,5), offers the following advantages for the assessment of DNA adducts ... 

Fig. 1. An immuno-slot blot fin the detection of 0 -hydro]Q thy]deoxygiianosine. Calf thymus DNA was hydrm ethylated in vitro with 6.6 mAf of hydroxyethyl-nitrosourea. Aliquots containing 300, 100, 33.3, 11.1, 3.7, 1.2, and 0 finol of O -hydroxyethyldeoxyguanosine in 3 (ig ofheat-denatured DNA (corresponding to 217-0 pmol/tnol of deoxyguanosine) were blotted onto nitrocellulose and incubated with a rabbit anti-O -hydroxyethyldeoiQ guanosine serum (NPZ-146-2,1 15,000 see ref. 4). Bound antibodies were reacted with a goat-antirabbit IgG horseradish peroxidase conjugate and detected with hydrogen peroxide and 4-chloro-l-naphth6l.

The immuno-slot-blot assay can be used to determine any heat or alkali stable DNA adduct for which a specific antibody has been raised. In ccmtrast to nonimmunological methods (HPLC, radiochromatography, -fX>sdabel-ing), the immuno-slot-blot requires that the DNA adduct of interest be known beforehand. A specific antibody must be available that does not show gnifi-cant crossreactivity with either normal or other modified DNA bases. 

This contribution will describe the use of the immuno-slot-blot assay for the quantitative assessment of promut enic fAalkyldeoxyguanosines and T-allq ldeoxyguanosines in the imidazole ring-opened form, as it is currently carried out in our laboratory. Briefly, alkylated DNA is denatured by heat or... 

Nitrocellulose filters, pore size 0.45 pm (BA 85), blotting papers (GB002-SB), and Minifold II Slot blot apparatus, all from Schleicher and Schuell, or equivalent. 

Depending on the crossreactivity of the antibody used, the sensitivity of the immuno-slot-blot assay may be increased by using sandwich-techniques for signal amplification, as used in immunohistochemistry, e.g., alkaline phosphatase-antialkaline phosphatase or peroxidase-antiperoxidase (seethisvol., Chapter 10). 

Nehls, P., Adamkiewicz, J, and Rajewsky, M. F. (1984) Immuno-slot-blot a highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA.y. Cancer Res Clin. Oncol 108, 23-29. 

Sahm K., MacGregor B. J., Jorgensen B. B., and Stahl D. A. (1999b) Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment. Environ. Microbiol. 1, 65-74. 

Gruen, L. C., McKimm-Breschkin, J. L., Caldwell, J. B., aad Nice, E. C. Affinity ranking of influenza neuraminidase mutants with monoclonal antibodies using an optical biosensor Comparison with ELISA and slot blot assays. J. Immunol. Methods., 1994, I6S, 91-100. 

---