Specificity versus binding

Add 1 ml of binding mix containing I-tPA in binding buffer in a final concentration of 5 nM into each well to allow binding to the cell surface pool of receptors. The binding is performed in either the absence or the presence of excess (1 fiM) unlabeled tPA for the determination of specific versus nonspecific binding. 

Mcl-l is a recognized major resistance factor in human cancer and Mcl-l over-expression has been linked to the pathogenesis of several cancers. Its anti-apoptotic properties are mechanistically related to its neutralizing interaction with BIM, BAK, NOXA and PUMA. ° Recently, a compelling Mcl-l BH3 stapled peptide has been reported to exhibit highly specificity and affinity to bind Mcl-l as well as effectively sensitizing cancer cells to caspase-dependent apoptosis in vitroJ An X-ray structure of Mcl-l complexed with this Mcl-l BH3 stapled peptide was also determined and showed inter-molecular contact of its hydrocarbon staple moiety with Mcl-l, accounting for the specificity versus other Bcl-2 family proteins. 

Protems can be physisorbed or covalently attached to mica. Another method is to innnobilise and orient them by specific binding to receptor-fiinctionalized planar lipid bilayers supported on the mica sheets [15]. These surfaces are then brought into contact in an aqueous electrolyte solution, while the pH and the ionic strength are varied. Corresponding variations in the force-versus-distance curve allow conclusions about protein confomiation and interaction to be drawn [99]. The local electrostatic potential of protein-covered surfaces can hence be detemiined with an accuracy of 5 mV. 

Figure 2. The binding and dissociation of FLPEP and receptor on intact neutrophils at 37 C The data are plotted as the specific binding of FLPEP (pmoles/10 cells) on a log plot versus time. Experimental details 10 cells/mL were exposed at time 0 to 1 nAf FLPEP. At 15, 30, 60, or 120 s, antibody to fluorescein is added to each sample. Fluorescence is monitored continuously during the additions. The data ate derived from a point-by-point comparison of the fluorescence measured under conditions of receptor binding and receptor blockade. Data are representative of observations in more than 10 separate experiments. (Reproduced with permission from reference 22. Copyright 19S7 Journal of Biological Chemistry.)...

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.

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