Dot blot technique

The mRN A coding for any particular enzyme can be determined by the dot blot technique, described in detail in Appendix b. The data take the form of photographic images on film. A dense image means that the cell or tissue extract, used... 

MeasiireTnent of bound DNA probe The total amount of DNA probe that is bound to the paper reflects the amount of mRNA coding for the protein of interest. The quantity of DNA probe that is bound to the paper can be measured because the DNA probe is radioactive, and no component of the tissue extract is radioactive. This is the dot blot technique. In cases where the various species of mRNA extracted from the tissue are separated from each other prior to transfer to the nitrocellulose, the technique is called the northern blot technique. Here, separation is by gel clectrophoresis. 

The dot blot technique takes advantage of the fact that the bases in DNA can also bind to bases in mRNA. Here, as before, A binds to T and G binds to C. The... 

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When the dot blot technique is used, as much as 1 mg of protein is placed in each well dot, and the total protein is tested for a visible immunogenic reaction. No visible dot means that the impurities are less than 5ppb. Lanes 1,4,6, and 8, contain Model Proteins A, A, B, and respectively. In Lanes 1,4,6, and 8, the top two dots contain Img, whereas the bottom two dots contain 0.1 mg thus the effect of a 10-fold dilution can be seen. Lanes 2 and 10 are the negative and the positive controls, respectively, showing the experiment to be working well. On further purification of Model Proteins A, A, B, and x, as shown in lanes 3,5,7, and 9, respectively, no spot can be detected. This means that purification of the model proteins has been obtained to the level where impurities detectable by the Western immunodotblot technique are less than 5 ppb. 

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). 

Antibodies may also be used to determine the presence or identity of soluble antigens by a process known generally as immunoblotting. In a technique known as dot blotting , soluble antigens are applied to a nitrocellulose membrane in the form of dots , antibody is then applied to the membrane, the membrane is washed to remove unbound antibody, and the presence or absence of the antigen is determined by similar means to that employed during immunohistochemistry. 

This technique, described in Section I, involves the construction of short DNA sequences (oligonucleotides) of approximately 20 bp that exactly complement either the mutated sequence or the normal sequence (hence, allele-specific ). The labeled oligonucleotide is then used to probe DNA from the individual in question. This DNA may be placed, for example, on a dot blot. Successful hybridization of the ASO containing the mutation indicates presence of the mutation, whereas hybridization of the normal ASO indicates presence of the normal sequence. Hybridization of both probes would be seen in a heterozygote. Because a different probe must be constructed for each mutation, this technique is practical when a limited number of mutations... 

If it is only necessary to check the immunoreactivity of an antibody towards a mixture of antigens, without attributing it to a particular protein, the antigen mixture (2-5 JU.L) can be spotted directly on the membrane. This technique, known as dot blotting (51) is useful for fast screenings of many antibodies simultaneously, for example in production of monoclonal antibodies. 

A simplified procedure in which protein samples are not separated electrophoretically but are spotted directly onto the membrane is called dot blot and is a good preliminary technique for the detection of an antigen in a sample. 

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]. 

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