In Western blotting

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

What concentration of primary antibody should be chosen for the initial experiments with a new antibody There is no clear answer to that, but if the optimal or suboptimal dilution is known from a Western blot, it often gives good results to start with this dilution, 10X and 100X less dilution in the initial experiment, and optimize further from there. Only in rare cases have we found that we should use less primary antibody than was used in Western blots. 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

The availability of synthetic peptides presenting an epitope that can be recognized by the primary antibodies used in Western blots allows their use as absolute standards when loaded in range of known amounts. Standard Western blotting procedures are performed on the sample containing the protein to be quantified along with a set of standards (known amounts of the epitope-bearing synthetic peptide). It is... 

Pyronin Y (Pyronin G, C.l. 45005) moves some faster than bromophenol blue and thus better indicates the electrophresis front. A further benefit is the strong binding to nitrocellulose which allows the identification of the electrophoresis front after immunochemicl reactions in Western blotting. 

Horseradish peroxidase catalyzes the cleavage of hydroperoxide substrates forming active oxygen, which oxidizes molecules resulting in a colored product. For application in Western blots the reaction product must be insoluble in aqueous buffer solutions. 

When AP will be taken for detection in Western blots, after antibody processing the membrane is equilibrated twice for 5 min each in Soln. A. Equilibration buffer is poured off and the color is developed in Soln. B completely covering the membrane. During this step the membrane should be moved very gendy only. 

In Western blots and in immunofluorescence, antisera often give unspecific reachons, especially when rabbit sera are used. These unspecifihes may be suppressed by pretreatment with liver powder. 

Liu, R. H. Jacob, J. Tennant, B. Chemiluminescent detection of protein molecular weight markers in western blot techniques. Biotechniques 1997, 22(4), 594-595. 

Even though primary and secondary antibodies are widely used in Western blotting detection systems, they do have some disadvantages. For proteins to be detected, specific antibodies must be available. It is often very time-consuming and expensive for a research laboratory to generate the proper antibodies if they are not available commercially. Even if antibodies are commercially available, they are very expensive. 

Define each of the following items in terms of their use in Western blotting. 

Sandhu, G.S., Eckloff, B.W.. and Kline, B.C. 1991. Chemiluminescent substrates increase sensitivity of antigen detection in Western blots. Bio-Techniques 11 14-16. 

Fig. 2. Gradient (4-20%) acrylamide gel of bone extracts. Two hundred micrograms of modem bone extract (M) and 400 /ig of three fossil bone extracts from the Archaic (7000 years b.p.) Windover site were stained with Coomassie Brilliant Blue. Products in the molecular weight range of albumin, IgG (heavy chain), and osteonectin are annotated, and the three proteins were identified in Western blots of this gel in both the modem and fossil bone extracts.

The classic LZ motifs common to all vertebrate dysbindin-1 orthologs (O Figure 2.2-5) are known to bind classic and hybrid LZ motifs of the same or different protein (Rodrigues and Park, 1993 Surks and Mendelsohn, 2003 Liu et al., 2006). We have seen no clear evidence in Western blots that dysbindin-1 ever forms homodimers, but it is capable of forming heterodimers with many of its known and candidatebinding partners with LZ (or hybrid LZ) motifs. Six of the nine established dysbindin-1 binding partners (i.e., 66%) listed in Q Table 2.2-6 have one or more LZ (or hybrid LZ) motifs, as do 7 out of 15... 

Using mlOCT-FP and PA31111, dysbindin-1 protein has been found in all tissues studied ( Figure 2.2-12a). It thus appears to be as ubiquitous as DTNBP1 gene expression (see above). In WESTERN blots, multiple bands can be seen, the exact number depending on species and tissue type ( Figure 2.2-12b and c). In human brain... 

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