Incubators hybridization

Vertical electrophoresis systems (Amersham Pharmacia Biotech, Piscataway, NJ, USA) Trans-Blot semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) hybridization incubator (Fisher, Hanover Park, IL, USA) personal densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA). Additional materials and equipment needed are described throughout the text. All chemicals are of reagent grade. 

Before prehybridization, nitrocellulose should be wetted with 1 X SSPE (or 1 X SSC), containing 0.1% SDS, whereas nylon membranes can be used directly. Prehybridization and hybridization is performed in polythene bags, plastic boxes (hybridization cassette) or hybridization incubators (Section 8.1.2). The cassettes and incubators are economical in the use of hybridization solutions. For plastic bags, use 0.25 ml/cm for nylon membranes and 0.1 ml/cm for nitrocellulose membranes of prefiltered (important ) prehybridization solutions. The bags should be massaged to completely distribute the solution and incubated with agitation. 

Dilute the antifluorescein/horseradish peroxidase conjugate 1000-fold in freshly prepared antibody binding buffer. The final volume should be at least equivalent to that used for hybridization. Incubate the blots in diluted conjugate with gentle agitation at room temperature for 60 min see Note 9). 

Incubate >1 /ig of RNA with a 3- to 10-fold molar excess of radio-labeled probe (about 150 to 600 pg per 10 /ig total RNA) in hybridization buffer. 

Close the hybridization chamber and place in a water bath that has been preheated to 42°. Incubate for at least 6 h. 

Fig. 13.7 Effect of MTX-LDH hybrid on cell proliferation (A) and viability (B) as determined by MTT assay and Trypan Blue exclusion, respectively. HOS cells were incubated with free MTX, MTX-LDH hybrid or LDH for 72 h.

Ironically, AP is the enzyme of choice for some applications due to its stability. Since it can withstand the moderately high temperatures associated with hybridization assays better than HRP, AP often is the enzyme of choice for labeling oligonucleotide probes. AP also is capable of maintaining enzymatic activity for extended periods of substrate development. Increased sensitivity can be realized in ELISA procedures by extending the substrate incubation time to hours and sometimes even days. These properties make AP the second most popular choice for antibody-enzyme conjugates (behind HRP), being used in almost 20 percent of all commercial enzyme-linked assays. 

Denature 1 pg of probe DNA (single stranded) with 5 pg of random hexanucleotide primers by boiling for 5 minutes and then rapidly chill on ice. Incubate at least 10 minutes to allow the primers to hybridize to random sites within the probe DNA. 

Since the site of modification on cytosine bases is at a hydrogen bonding position in double helix formation, the degree of bisulfite derivatization should be carefully controlled. Reaction conditions such as pH, diamine concentration, and incubation time and temperature affect the yield and type of products formed during the transamination process. At low concentrations of diamine, deamination and uracil formation dramatically exceed transamination. At high concentrations of diamine (3M), transamination can approach 100 percent yield (Draper and Gold, 1980). Ideally, only about 30-40 bases should be modified per 1,000 bases to assure hybridization ability after derivatization. 

In a DNA array, gene-specific probes are created and immobilized on a chip (silicon wafer, nylon or glass array substrate). Biological samples are labeled with fluorescent dyes or radioactivity. These labeled samples are then incubated with the probes to allow hybridizations to take place in a high fidelity manner. After incubation, non-hybridized samples are washed away and spot fluorescent or radioactivity signals resulting from hybridization can be detected. 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

Figure 4.2 Generalized outline of a gene chip. In this example, short oligonucleotide sequences are attached to the anchoring surface (only the outer rows are shown). Each probe displays a different nucleotide sequence, and the sequences used are usually based upon genome sequence information. The sequence of one such probe is shown as AGGCA. By incubating the chip with, for example, total cellular mRNA under appropriate conditions, any mRNA with a complementary sequence (UCCGU in the case of the probe sequence shown) will hybridize with the probes. In reality, probes will have longer sequences than the one shown above...

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

Incubate for 2 h in the vapor-tight container in a droplet of IX SSC at the hybridization temperature. 

Figure 13.15 Detection of a specific sequence of DNA by hybridization to a 32P-labelled cDNA probe. DNA is transferred to nitrocellulose and incubated with the probe. After washing, specific binding is visualized by autoradiography. The DNA sequence detected by the probe is present in lanes 2, 3 and 5 but not 1 and 4.

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

The radioactive-labeled sample see Note 5) is now denatured by incubating it in a boiling (95-100°C) water bath for 2 min and then transferring it on ice for 2 min. The denatured probe is combined with 15mL prewarmed hybridization solution in a disposable 50- or 15-mL plastic tube and carefully mixed together. 

The hybridization phase entails thermal denaturation of double-stranded DNA and incubation of the probe with the denatured DNA at a temperature 25°C below the melt temperature. Unless one is interested in partial homology, lower temperatures should be avoided. However, addition of formamide promotes the hybridization, thereby permitting the use of a lower temperature, if it should be required, to prevent structural modification. 

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