Immunoblot Detection

IMMUNOPROBING WITH DIRECTLY CONJUGATED SECONDARY ANTIBODY 

Blocking buffer (see recipe) appropriate for membrane and detection protocol 

TTBS (nitrocellulose or PVDF) or TBS (neutral or positively charged nylon see recipes for both solutions) 

Secondary antibody conjugate horseradish peroxidase (HRP)- or alkaline 

Heat-sealable plastic bags Powder-free gloves Plastic box 

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Western blotting—used to identify a specific protein or group of proteins by immunoblotting (detection by antibodies). 

Figure 10.13. Comparison of immunoperoxidase (Lane A), peroxidase anti-peroxidase (Lane B) and biotin-streptavidin (Lane C) immunoblotting detection methods.15 [Reprinted, with permission, from K. E. Johansson, in Handbook of Immunoblotting of Proteins , O. J. Bjerrum and N. H. H. Heegaard, Eds., CRC Press, Boca Raton, FL, 1988. 1988 by CRC Press, Inc.]...

Cooper, P.S., W.K. Vogelbein and P.A. Van Veld. Immunohistochemical and immunoblot detection of P-glycoprotein in normal and neoplastic fish liver. In Techniques in Aquatic Toxicology, edited by G.K. Ostrander, Boca Raton, EL, CRC Press, pp. 307—325, 1996. 

Matson, C.T. and Drewes, L.R. (2003) Immunoblot detection of brain vascular proteins. Methods in Molecular Medicine, 89. 479-487. 

The protein A acyl carrier protein-I gene was partially sequenced and found to be completely homologous to our desired construction (7). The purified protein from E. coli N4830 cells was approximately 90% pure as judged from SDS PAGE using both Coomassie staining and Western immunoblot detection (data not shown). This purified preparation was stable at -20° for several months and was used as source of fusion AGP for in vitro enzymatic characterizations. 

V. Bioluminescence-enhanced assay. This assay, originally developed for im-munoblot detection (20), utilizes coupled enzyme reactions initiated by APase that releases D-luciferin from D-luciferin O-phosphate (Novabiochem AG). The D-luciferin subsequently reacts with ATP and oxygen in a reaction catalyzed by luciferase (from firefly Photinus pyralis), resulting in light emission that can be detected on a sensitive photographic film (see Section D, Chapter 8). The reaction is performed in a pH 8.0 buffer. In immunoblot detections, this assay achieves a sensitivity of 5—50 pg protein (or 2—20 x 10 IgG molecules). 

Laboratory confirmation is vital to effective treatment of HSV, especially in individuals in whom a clinical diagnosis cannot be obtained. There are several methods by which a definitive diagnosis may be acquired, and these include virologic typing, serologic diagnosis, rapid point-of-care antigen detection, enzyme-linked immunosorbent assay (ELISA), immunoblot, and DNA polymerase chain reaction.27... 

The practicing clinician has a number of different tests available to aid in the evaluation of patients with suspected hepatitis C. These include measurement of alanine aminotransferase (ALT) levels, liver biopsy, serological tests (ELISA and recombinant immunoblot assay), and molecular methods for detection and quantitation of HCV RNA. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

Immunoblot analysis is conducted with antibodies to the His8-tag epitope (to detect the TIF32-His8 subunit Santa Cruz) or with antibodies to the other factors. 

A significant advantage of this method is that is provides quantitative information since the IEF technique actually separates the phospho- and dephospho- forms of the protein, the ratio of these species as detected by IEF/immunoblotting is a direct readout of the protein s phosphorylation status within the cell (assuming no selective loss of one species during isolation and analysis). 

The criteria we used for inclusion of a mutation in this section are (1) that binding or uptake was detected or the presence of transporter in the membrane was confirmed by immunoblotting or immunos-taining, and (2) mutation must have produced a decrease in apparent affinity for substrates or affinity for... 

Actually doing tissue printing is the best way to understand its simplicity (Fig. 1). Wide variations in the technique are possible and can be developed easily and quickly according to need. The following protocol for using tissue prints to detect tissue distribution of IAA-protein con-juguates in okra fruit details the immunoblotting technique as follows. 

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