Proteins capture

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

Bead-bound PF-4540124 (2) was incubated with the soluble fraction of a mouse lung homogenate in the presence and absence of 1 (100 J.M). The type of protein captured was restricted by having 1 mM ADP and GDP in both samples. The beads were pelleted and washed before being treated (both samples) with 1 (100 iM) to elute specifically bound proteins. 

Figure 14 6 Silver-stained SDS-PAGE gel of PatA binding proteins. Lane 1, sample 1 nonspecific proteins captured by the streptavidin-agarose resin Lane 2, sample 2 proteins affinity captured by the presence of B-Pat A Lane 3, sample 3 affinity capture of target proteins was blocked by prior addition of free PatA before incubation with B-PatA. The two arrows point to two proteins specifically detected in sample 2 versus sample 1, which were also lost due to competition in sample 3, with apparent molecular weights of 38 and 48 kDa.

Nishino H, Huang CS, Shea KJ. Selective protein capture by epitope imprinting. Angew Chem Int Ed 2006 45 2392-2396. 

The most well-known approach of using recognition elements in protein arrays was developed by Ciphergen Biosystems (Fremont, CA). This company offers a number of customized surface chips for protein capture to be used prior to MALDI-TOFF analysis. 

There are several possible arrangements tolerating the presence of particles during adsorption of proteins to particulate matrices. Batch adsorption in stirred tanks is performed by contacting adsorbent particles with a cell containing suspension. After protein capture the adsorbent is separated from the broth and the protein of interest can be eluted. This procedure has been described for the isolation of antibiotics [12], the purification of ot-amylase from B. amylo-liquefaciens broth [13], and the isolation of the prothrombin complex from... 

Different extraction techniques have been used for the characterization of the artichoke s proteome by CPLL. The initial extraction buffer contained 50 mM Tris-HCl (pH 7.4), 50 mM sodium chloride, 2% (m/v) CHAPS, 1% (m/v) sodium dodecyl sulfate, and 25 mM dithiothreitol. Protease inhibitor cocktails were added to the extraction buffers to prevent the action of protease. The first extract was then diluted 1 10 (v/v) with the same buffer without sodium dodecyl sulfate to facilitate the protein capture. The extract was separated into four equal aliquots each of them was titrated to different pH values (4.0, 7.2, and 9.3). The pH of the fourth aliquot was reduced to 2.2 with addition of 0.1% TFA and formic acid. Then individually, each aliquot was added with 100 pL of CPLL beads overnight at room temperature under gentle shaking. 

As a summary, in almond s milk, 137 unique protein species were found, whereas in the case of orgeat syrup (bitter almond extract), 13 proteins could be identified. Nevertheless, both deep proteome analyses could assess the genuineness of these products. This was not necessarily the case for cheap orgeat extracts sold in supermarkets, suggesting a possible production complemented with synthetic aromas. When present, proteins of this investigation could be easily visible by SDS-polyacrylamide gel electrophoresis after protein capture at pH 7.0 and 9.3 (65). 

The production examples described here, and the information published since this chapter was written, clearly indicate that the expanded-bed operation offers an efficient alternative to the conventional protein capturing process. 

In the so-called direct assay format, a biotinylated component binds to an antibody directly coupled to acceptor beads or to a protein captured by this antibody (Figure 8.4A). In the indirect assay format, the antibody used for capturing the biomolecule is in turn bound by a secondary antibody or by Protein A conjugated to the acceptor beads. In principle, any method capable of capturing the interaction partners to donor and acceptor beads, respectively, is suitable for setting up an AlphaScreen assay. 

One such method is fhe microsphere-based xMap technology (Luminex, Austin, Texas, USA), which is a multiplex flow cytometric method based on antibodies coupled to spectrally specific fluorescent microspheres (Vignali, 2000). Another fluorescent reporter antibody binds the protein captured on the microspheres. 

The controls are important in this work and should include an antibody-free condition to allow for proteins that bind directly to the bead and a lysate-free sample to account for the protein capture system employed. The optimum conditions can be considered to be those that give the maximum number of specific (found in the test but not control) bands. Consideration should be given to the number of nonspecific proteins, as these may make it difficult to identify the specific ones. 

Protein beads, chips, and/or other affinity-based supports represent an important emerging technology for the separation of proteins from complex mixtures and/or for the examination of specific protein-protein interactions. For example, protein chips consist of arrays of small spots on a solid support (e.g., aluminum, glass, etc. plate), with each spot containing a protein capture moiety or bait, either chemical... 

Yoshitani N, Saito K, Saikawa W, Asanuma M, Yokoyama S, Hirotal H (2007) NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance. Proteomics 7(494) 9... 

MIP-based assay and the MIP sensor chip was able to be regenerated many more times than the antibody sensor chip. In another application reviewed by Nedelkov and Nelson [ 173], the detection and quantitation abilities of SPR technology were coupled with mass spectral identification of proteins captured, either directly on the sensor chip or following elution from the chip surface. 

Micelles have been used to build lipid bilayers around membrane proteins captured on sensor surface to mimic their natural environment. A good example of this type of application involves the study of G-protein coupled receptors (GPRCs). Receptor was captured on an LI chip surface using an immobilized antibody that recognizes an additional tag presented on the receptor followed by injection of micelles to form the hpid bilayer [19] (Fig. 12). Others have used this approach to monitor interactions of hg-ands and G-proteins with GPRCs [20]. This approach may provide a general method for studying a variety of membrane-associated systems and even ion channels. 

T. Kodadek, Transformation of low-affinity lead compounds into high-affinity protein capture agents, Chem. Biol. 2004, 11, 1127-1137. 

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