Incubation Step

In practice, including blocking reagents in all incubation and rinse solutions insure that the tissue is adequately blocked. It is recommended that the blocking regents be added to all subsequent incubation steps, and if possible, to the rinse steps between incubations. 

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The second patent describes the use of a microbial mixed culture (Hansenula sydowiorum, Hansenula ciferrii, Hansenula lynferdii, and/or Cryptococcus albidus) in coal desulfurization [160], In this process, the raw mined coal is ground to a particle size smaller than 200 mesh forming a slurry with water, at a solids concentration of less than 40wt%. The bacterial cultures are then inoculated into the feedstock slurry. An incubation step is carried out at a temperature near 25°C and at a pH close to neutral. The highest removal achieved was in the range of 46% S removal. 

Transfer 600 p1 of this mixture to another micro tube containing 10 /(I of antisense ODN (10 pmole/p ) complementary to the cleavage site on the mRNA. The remaining 70 to 100 p serves as a control sample ( uncut ) and will be subjected to the following incubations (steps 3 to 6). 

Stain the gel with silver using standard procedures. Careful examination of the gel should reveal bands that appear in sample 2 and not sample 1, which can be assumed to be captured specifically by the biotin-conjugate. Furthermore, if these same bands are not present, or are lower in intensity in sample 3, they can be assumed to be specifically captured in sample 2, but lost in sample 3 due to occupation of the binding-site by the free compound during the pre-incubation step. 

Figure 3 Layout of competitive solid-phase antibody immunoassay format in an incubation step antigen (Ag) and labeled antigen (Ag-L) compete for the solid-phase antibodybinding sites (Ab) after the solid-phase antibodies are washed, the activity of the bound labeled antigen is measured, and a calibration curve constructed. The measured signal is inversely proportional to the antigen concentration.

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). 

Depending on the starting material, preantibody incubation steps may vary and are outlined in Chapters 9-12. The following assay begins with the removal of the slides from the overnight 10% normal serum incubation step. Since the ABC technique is universal in application, and a biotinylated secondary antibody exists for virtually any primary antibody, this protocol will assume a monoclonal assay and a primary antibody from a mouse hybridoma. A good secondary antibody to use in this situation is a biotinylated antimouse antibody made in horse. Therefore, the overnight serum incubation in this case would have been with 10% normal horse serum in PBS (see Note 4). 

If the antigen of interest is abundant and additional sensitivity is not required, then a two-step direct method, rather than a three-step indirect method, can be anployed. With the two-step method, the primary antibody is a biotin-labeled mouse monoclonal antibody, which is followed directly by the ABC incubation step. Because of the abundance of human immunoglobulins in tissue sections, methods for staining immunoglobulin often employ biotin-labeled mouse antihuman immunoglobulin reagents. 

Another antibody-based immunosorbent assay that can be used to determine HAT activity uses a secondary anti-IgG antibody, which is directed against the primary antibody and is labeled with the lanthanide Europium (Eu). After another washing step that removes all nonbound secondary antibody, one last incubation step is performed, which releases the lanthanide ion from the antibody, so that the final detection of time-resolved fluorescence (340/615 nm) caused by the released metal ion correlates with the acetylation level of the oligepeptide histone substrate, which is correlated with enzymatic activity. So far. 

Wash the hlot after the antibody and conjugation incubation steps with Soln. A, followed by an equilibration in 2 ml/cm of Soln. B for 2-3 min. 

To demonstrate that the mixture does not manifest antimicrobial activity, carry out the test as described previously up to the incubation step and add an inoculum of viable cells of the specified aerobic bacteria, anaerobic bacteria, and fungi. 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

On completion of the two pre-incubation steps, 10 pi of the preincubated sample will be added to 500 pi of the preincubated emulsion in triplicate, and the LPL-cata-lyzed triglyceride hydrolysis will be allowed to proceed for 1 h at 28°C. The reaction is stopped by adding 5.33 ml of methanol chloroform heptane (56 50 4 by volume) and vigorous shaking. 

Perform all subsequent steps at room temperature. Keep plates in the humidified box during incubation steps. 

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