Dot blotting

In a preliminary report, Ross et al. [40] used affinity chromatography to identify a putative bovine renal brush border Na /H exchanger. Brush border membranes were solubilized with Triton X-100 and chromatographed sequentially over lentil lectin Sepharose 4B and 5-(A-benzyl-iV-ethyl)amiloride coupled to epoxy-activated Sepharose 6B. The eluant contained 178- and 146-kDa proteins that were susceptible to Endo-F. Moreover, the eluants reacted on dot blot immunoassays with antisera to a 20-amino acid peptide of a human Na /H exchanger vide infra). The relationship between these proteins and the 66-kDa protein previously identified by the same investigators using amiloride photolabeling is presently unclear. 

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

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). 

Figure 6. 125I-F-D Heparin dot blot assay. See text for methods and abbreviations. Upper panel incubation with 125I-F-D labelled porcine mucosal heparin alone, Lower panel the same conditions, but a 100-fold excess of unlabelled heparin has been added to the labelled heparin.

Rupp and Locker3 Rat liver Homogenized in a solution containing SDS, and proteinase K Not available, using hybridization including dot-blot method RNA purified from FFPE tissue is suitable for hybridization. 

Transfer proteins onto a nitrocellulose membrane using any appropriate procedure, including dot blotting the protein solution onto the surface. 

Oser, A., Roth, W.K., and Valet, G. (1988) Sensitive non-radioactive dot-blot hybridization using DNA probes labeled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acids Res. 16, 1181-1196. 

Quantification of the DNA isolated from the product involves concurrent inclusion in the dot blot assay of a set of spots, containing known quantities of DNA, and being derived from the producer cell. After autoradiography, the intensity of the test spot is compared with the standards. 

An alternative assay format entails the use of virus-specific DNA probes. These can be used to screen the biopharmaceutical product for the presence of viral DNA. The assay strategy is similar to the dot blot assays used to detect host-cell-derived DNA contaminants, as discussed earlier. 

PAMAM dendrimers or modified PAMAM dendrimers (with oxiamine or sulfhydryl surface functionalities) exclusive of one another. These antisera recognize den-drimers in ELISA (enzyme-linked immunosorbent assays), dot blots and Western blots. The immunogenicity of dendrimer-protein conjugates has implications for therapeutic use of dendrimers as vaccines and we anticipate that antidendrimer antibodies will have applications in patterning and assembling nanostructures containing dendrimers. 

Immune detection is a key utility of antibodies in biotechnology [3, 5]. Antiden-drimer sera efficiently detect dendrimers in multiple assay formats, including enzyme-linked immunosorbent assays (ELISA), and in Western and dot blots [3, 5], ELISA assays are commonly used to quantitate proteins, and a quantitative ELISA could be developed for dendrimers using our sera, though doing so would require development of dendrimer standards of known concentration that could be used for calibration. 

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. 

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. 

ICC of astrocyte cell culture shows CYP2E1 over cytoplasm and processes but more pronounced over nuclear membrane immunogold labeling agrees. Ethanol causes an increase in CYP2E1 as determined by ICC and Dot blot (Montoliu et al., 1995). 

Allde-spedflc ohgonudeotides (ASO) are relatively short DNA probes that under stringent conditions can differentiate between alleles of a gene. To design an ASO, one must know the mutation involved in the disease. An ASO is most usefiil if it is specific for the particular mutation that accounts for most cases of the disease. They are usually used to probe dot blots. 

Detection of amplified DNA by expected size (gel electrophoresis) or by sequence Oprobing a dot blot)... 

RFLP analysis of the p-globin gene for genetic testing has been replaced, by PCR In combination with ASO probes on dot blots. The blot shown here corresponds to the family whose pedigree is shown in Figure T7-9. In the mutant allele, glutamate (E) at codon 6 is replaced by valine (V). 

When the amplified material was tested under stringent conditions using aUele-specific probes, the results indicated that the child was heterozygous at tiie p-globin locus. Which dot blot shown below best represents the results from this family ... 

B. Flanking the CAG repeat followed by a dot-blot using a CAG repeat-specific DNA probe. 

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... 

For additional discussion of allele-specific oligonucleotide (ASO) probes and dot blots, see Section I, Chapter 7 Genetic Testing. 

DNA adsorption properties were first studied using a variety of solid supports for classical analysis methods including Southern and Northern transfers, dot-blotting, colony hybridization and plaque-lifts [31,32]. Studies of the interactions between nucleic acids and nitrocellulose revealed that molecular weight, finite macromolecular conformation, ionic forces and weaker forces of attraction all play a role. DNA is retained on nitrocellulose only in... 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

The advent of the polymerase chain reaction (PCR) made it possible to directly spot down cDNA amplicons onto membranes, giving rise to the Southern dot blot format. In fact, the dot blot should be regarded as one of the earliest, if not the first, array format (albeit a macroarray). Why did many abandon the membrane in favor of the glass substrate for DNA microarrays ... 

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