Reverse blot assays

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

The amplicons prepared are subsequently analyzed by a variety of methods. They can be hybridized to oligonucleotide probes in reverse or forward dot-blot assays. The panel of probes is chosen to cover critical polymorphic positions in the HLA gene tested. The pattern of reactivity of the panel can then be analyzed to assign allele identities. ... 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

Fig. 3. Essential fatty acid deficiency-induced epidermal hyperproliferation and protein kinase C (PKC)-P activity are suppressed by dietary linoleic acid. Membrane-associated epidermal PKC isozyme (a and p) activities were determined in the epidermal extracts from control, essential fatty acid-deficient (EFAD) and reversed guinea pigs (EFAD guinea pigs restored to normal). Specifically, epidermal high-speed particulate membrane fractions were prepared and PKC isozyme activities from each dietary group were assayed as described previously (16). The upper portion of the figure represents PKC-p and PKC-a activities of the three dietary groups values are means SD (n = 12) from three separate experiments. The lower portion of the figure represents the expression of the 2 PKC isozymes. To determine PKC-P and PKC-a expression, 30 mg protein of solubilized epidermal membrane preparation from each dietary group was subjected to gel electrophoresis (SDS-PACE, 10 % gel) followed by Western blot assay with specific PKC-a and PKC-p. The gel electrophoresis data were reproducible in three separate experiments.

Ripe tomato fruits accumulate significant amounts of lycopene, but only trace amounts of xanthophylls. Dharmapuri and others (2002) overexpressed the lycopene (3-cyclase (b-Lcy) and (3-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter, and transgene and protein expression was followed through semiquantitative reverse- transcription PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of (3-carotene, (3-cryptoxanthin, and zeaxanthin the carotenoid composition of leaves remained unaltered, and the transgenes and the phenotype were inherited in a dominant Mendelian fashion. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

NIS mRNA such as by northern blotting. Also, through the process of reverse transcription and a polymerase chain reaction (hereafter referred to as RT-PCR), a DNA strand identical to the RNA strand is formed and detected in a very sensitive assay. These RNA-based assays are two methods that can determine whether the symporter mRNA was present in a given tissue. Detection of symporter mRNA would indicate that the gene was being expressed. 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

In order to establish that a process results in consistent product, analytical tests should be established and qualified by using them to test a reference standard. These tests are developed as in-process test methods to track the quantity and quality of product during optimization of the purification process. In-process analyses often involve using ion exchange HPLC, reverse phase HPLC, SDS-PAGE, Western blot, and/or ELISA assays. 

Table 2. DMRTl expression in vertebrates. GSD-genetic sex determination, TDS-temperature dependence sex determination, T-testis/genital ridge in male embryo, O-ovary/ genital ridge in female embryo, K-kidney, L-liver, H-heart, M-muscle, LU-lung, S-spleen, ISH-in situ hybridisation, RT-PCR-reverse transcription-polymerase chain reaction, qRT-PCR-quantitative RT-PCR, NB-Northern blot, DB-Dot blot, IHC-immunohistochemistry, WB- Western blot, RPA-RNase protection assay.

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