Affinity capture

Fig. 9 Purification of ELPs by ITC is based on the reversible inverse phase transition. Le/i Protein purification via direct ELP fusions. A soluble ELP fused to a target protein becomes reversibly insoluble upon increasing temperature above 7,. Center Protein purification via ELP coaggregation. An excess of free ELPs enhances the aggregation of trace quantities of ELP-fusions. Right Purification via ELP-mediated affinity capture (EMAC). ELPs are fused to capture proteins, which bind specifically and reversibly to a target protein. This target protein can then be aggregated at temperatures above the T,. Adapted from [38] with permission from Elsevier, copyright 2010...

The third purification procedure is based on the combination of temperature-triggered aggregation and affinity capture and has been used to not only purify proteins [50-54] but also other molecules [55, 56], and for the removal of pollutants from a solution [57-59]. For this procedure, ELP is conjugated to a capture reagent, which can be done either genetically or chemically. This procedure eliminates the need for cleavage of ELP after purification and introduces the potential to recycle the ELP. 

Bundy, J. Fenselau, C. Lectin-based affinity capture for MALDI-MS analysis of bacteria. Anal. Chem. 1999, 71,1460-1463. 

The high potentials required for electrospray show that air at atmospheric pressure is not only a convenient, but also a very suitable ambient gas for ES, particularly when solvents with high surface tension, like water, are to be subjected to electrospray. The oxygen in the air, which has a positive electron affinity, captures free electrons and acts as a discharge suppressor. 

The second group identified 41, which inhibited Wnt pathway activity and proliferation of cancer cells with an aberrant Wnt pathway at micromolar concentrations in contrast, the structurally related analog 42 had no effect. Immobilization of 41 and affinity capture followed by proteo-mics identified several potential targets, including TANK-1 and TANK-2,... 

Affinity capture of PatA-binding proteins using... 

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.

The techniques developed to study protein interactions can be divided into a number of major categories (Table 31.1), including bioconjugation, protein interaction mapping, affinity capture, two-hybrid techniques, protein probing, and instrumental analysis (i.e., NMR, crystallography, mass spectrometry, and surface plasmon resonance). Many of these methods are dependent on the use of an initial bioconjugation step to discern key information on protein interaction partners. 

Turecek, F. (2002) Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis. J. Mass Spectrom. 37, 1-14. 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) is another recently introduced technique for quantification of proteins, and to date has most often been applied to clinical enzymology.60 The product conjugates of the enzymatic reaction between the synthetic substrate and targeted enzyme are captured by immobilized affinity reagents, purified, released into solution, and analyzed by ESI-MS. 

Among the techniques listed in Section 1.2.1, the two most documented approaches in addition to SPE, LLE, and PPT are solid phase microextraction (SPME) and affinity capture of analytes based on molecularly imprinted polymers (MIPs). Recent developments in these areas are briefly discussed below. 

Staining patterns of two-dimensional gels show the six RC ATPases to be present at comparable levels, and affinity capture of yeast 26S proteasomes indicate the presence of one copy of each in the regulatory complex. Mutational analysis in... 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology... 

In this strategy, phages are affinity-captured on immobilized substrate under conditions in which the enzyme is inactive, for example, in the absence of an essential metal ion or a cofactor. Active phage-enzymes are then eluted by triggering catalysis by addition of the metal ion or cofactor, taking advantage of the lower affinity for the product than for the substrate. 

As shown in Figure 6.1, the protocol starts with labeling the phages with the substrate. Several approaches have been used for this labeling step [11,13,14]. The active enzymes are then turning over the substrate into product by intraphage catalysis. Finally, the pro duct-labeled phages are selected by classical affinity capture. 

In this strategy, it is very important to avoid interphage catalysis. Therefore, the phage concentration should be kept low (lower than 10-9 M) and phages should not be precipitated with PEG. Because removal of excess label is generally required before affinity capture, it should be done by phage dialysis or size-exclusion chromatography. 

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