Western blotting primary antibodies

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. 

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... 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

The binding of an antibody to its antigen has also been utilized in the detection of specific proteins on a solid support, such as a western blot, where size-separated proteins are immobilized on a nitrocellulose or nylon membrane (see Section V.B). In this case, the primary... 

Detecting exposure to HIV ELISA assays and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient s blood sample. ELISA assays are used as the primary screening tool, because they are very sensitive. These assays sometimes give false-posi-tives, however, so western blots, which are more specific, are often used as a confirmatory test (Figure 32.23). [Note ELISA and western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]... 

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. 

El 2. Is the detection system used in this experiment, treatment with a single identifying protein, Con A-HRP, as specific as a traditional Western blot using two proteins (a primary antibody and a secondary antibody) Why or why not ... 

No. Two highly specific interactions between antibody and protein occur in the traditional Western blot, which uses a primary and secondary antibody. With Con A-HRP, only one specific interaction takes place. Only a specific class of proteins is identified in this experiment, not a specific protein. 

Different investigators incubate blots with primary antibodies for different lengths of time (reviewed in ref. 3) Preliminary studies with some of our antibodies have revealed that the signal intensity on Western blots is greater when blots are incubated with antibody overnight rather than 1-2 h at room temperature (G. Humphrey and S H. K, unpublished observations)... 

Diluted antibody solutions can be reused multiple times They should be stored at 4°C after additional aliquots of penicillin/streptomycin and sodium azide have been added Some workers believe that the amount of nonspecific (background) staining on Western blots diminishes as antibody solutions are reutilized Antibody solutions are discarded or supplemented with additional antibody when the intensity of the specific signal begins to diminish 10. Choice of wash buffer after incubation with primary antibody 2M urea is included m the suggested wash buffer to diminish nonspecific binding. Alternatively, some... 

Determine the approximate expression level of soluble BCCP fusion by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) together with western blot analyses (18) using an anti-His primary antibody (see Note 5). 

To determine the extent of biotinylation of the BCCP fusion protein, cany out a supershift Western blot assay (again with an anti-His primary antibody) in which equivalent crude lysate samples are pre-incubated with or without streptavidin (0.1 g/mL) (Fig. 3 see Note 6). 

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