Non-specific binding

Both domains of the POU region bind to DNA by the usual combination of non specific binding to the DNA backbone and specific binding to the bases. The contacts between the homeodomain and DNA are similar to those of the engrailed homeodomain (compare Figures 9.10b and 9.15a) and the... 

NSB (non-specific binding) tubes These tubes contain all reagents except the binding protein. They will therefore show those counts that cause an error in that they are due to free label being counted as bound. 

Important criteria for biomolecule immobilization are high functionality of the chip surface high immobilization capacity optimized density of functional groups stable linkage between the biomolecule and the solid support good accessibility for the interacting molecules low non-specific binding. 

P.W. Robertson, L.R. Whybin, and J. Cox, Reduction in non-specific binding in enzyme immunoassays using casein hydrolysate in serum diluents. J. Immunol. Methods 76, 195—197 (1985). 

H.C.B. Graves, The effect of surface-charge on non-specific binding of rabbit immunoglobulin-G in solid-phase immunoassays. J. Immunol. Methods 111, 157—166 (1988). 

For reasonable conditions [B] should be below the expected KD value, and [A] should span a range from 0.1 -10 times KD. Analysis of real binding curves has to consider additional sources of error such as equilibrium not being achieved, errors in the determination of the concentrations, or non-specific binding. 

A system based on microdialysis coupled with flow-injection chemiluminescence detection allows for direct sampling of unbound drug without extractive sample preparation [72], A similar approach based on continuous ultrafiltration has also been reported [73]. Modifications designed to overcome challenges of low solubility and high-non-specific binding in the ultrafiltration approach have also been described [74]. 

Fc receptors present on the cell membrane of macrophages, monocytes, granulocytes, lymphocytes, and some other cells may, in theory, non-specifically bind Fc portion of antibodies used for immunolabeling. Typically, blocking endogenous Fc receptors involves using normal serum derived from the same species used to produce the secondary antibody. To block endogenous Fc receptors, incubate sections for 15 30 min with normal serum (2 5% v/v) from the host species of the secondary antibody. 

Diluted antiserum is added to all tubes except the non-specific-binding control specimen to which buffer is added,... 

A third issue arises with NCEs that bind to plastic surfaces due to the hydrophobic physical nature of the molecule. A significant loss due to this type of non-specific binding to the Caco-2 device may lead to underestimation of permeability. Addition of protein (e.g., bovine serum albumin) in the receiver compartment appears to be capable of preventing some non-specific binding [23],... 

Dai H, Meyer M, Stepaniants S, Ziman M, Stoughton R. 2002. Use of hybridization kinetics for differentiating specific from non-specific binding to oligonucleotide microarrays. Nucleic Acids Res 30 e86. 

The Use of Non-specific Binding To Overcome Limitations of Crude Templates... 

The coupling of the GPC spin column/ESI-MS screening results with NMR (2D HSQC) is a powerful method for confirming that the non-covalent binders identified by the MS experiments truly bind at the predicted active site by observing NMR chemical shift perturbations in the vicinity of the protein active site [1, 15]. In contrast, the absence of chemical shift perturbations or a random distribution of chemical shift changes on the protein surface would imply a lack of an interaction of the compound with the protein or potentially the existence of non-specific binding. The development of the GPC spin column/MS/NMR assay... 

ALIS reports only compounds that bind directly to the target of interest, preventing false positives that arise from off-target activity or interactions with substrates or other reagents. Since ALIS directly identifies bound components by MS, the incidence of false positives based on bulk effects and non-specific binding is lower than that of biochemical assays that yield a secondary readout of activity. 

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