Unspecific binding

Some of the polyclonal antibodies or the antibodies produced by the animal before the immunization might cause unspecific bindings. To control this, it is important to obtain a preimmune bleed from the same animal. The preimmune serum should also be used as a guide in the initial experiments to determine the dilution of the antibody, and later on... 

Preimmune serum instead of the primary antibody. If positive, it indicates that unspecific binding has taken place. Try again with a higher dilution of preimmune serum and antibody. 

Omission of primary antibody. If positive, it shows unspecific binding of secondary antibodies. Dilute or try another secondary antibody. 

Definition of the background activity of microinjected oocytes is a clear prerequisite for any functional cloning procedure, as a positive signal needs to be distinguished from the endogenous transport activities. Key characteristics of the carrier, such as a dependency on sodium ions or a specific inhibitor are very helpful. In addition, unspecific binding of the radioactive transporter substrate to the oocyte membrane needs to be examined carefully. 

Soak GF/F filters (diameter 1.3 cm, Whatman) and a sheet of filter paper (GB002, 102x133 mm, Schleicher and Schuell) for 1 h in incubation buffer containing 1% Sigmacote to reduce unspecific binding of the RNA molecules to the filters. 

Determination of unspecific binding reaction by competition of the RNA aptamers with cocaine 79 pi incubation buffer, 10 pi nAChR-enriched plasma membranes (800 pg/ml protein 1.6 pM receptor), 1 pi P-RNA dilution containing 10 mg/ml t-RNA anti-RNAse (40 U/pl), and 10 pi cocaine (10 mM). The percentage of binding of the ( P) RNA aptamers to the... 

To reduce unspecific binding, mix a 200-pl aliquot of the clear supernatant with 2 pi pre-immune serum or unspecific antibody and a further 200 pi aliquot with 50 pi precipitation aid. Rock at 0 °C for 1 h and spin at 1000 x g. Transfer the supernatant into a fresh container and fill it up to 1000 pi with Soln. A. Add 0.5 - 5 pi of the specific antiserum and monoclonal antibody, respectively, and incubate on ice for 1 h. Prepare a second sample containing pre-immune serum instead of antiserum. 

Flavonoids, as food components or potential drugs, interact with a wide range of proteins by distinct mechanisms weak and rather unspecific binding of tannins to proline-rich or histi-dine-rich random coils leading to protein precipitation, specific enzyme inhibition, and... 

Nevertheless, the major issue in the miniaturization of this kind of assays continues to be the strong non-selective interactions with the polymers. This problem is often avoided, or at least minimized, on large scale experiments such as chromatographic separations. However, it is not easy to solve in the case of microsensors or microchips and direct or indirect assays, where competition can be strongly affected by unspecific binding. 

A common analytical problem in immunoassays is that of cross-reactivity, which is the unspecific binding of the therapeutic antibodies determined in the assay with a second (or more) antigen(s). These interfering bindings must be kept to a minimum. Among other factors, specificity of the enzyme-labeled anti-Fc and anti-Fab antibodies used for detection is of major importance. In general, each assay design bears its special drawbacks that must be carefully evaluated for its intended use. 

Blocking To decrease unspecific binding treatment with blocking buffer I or blocking buffer II is necessary (see Note 2). Blocking should be carried out for at least 2 h at room temperature. Treatment overnight at room temperature or over the weekend at 4°C is also possible. 

After incubation, if a spot signal also shows a corresponding spot signal by reflection on a virtual diagonal (Pos.1,1 to pos. x,x), it is possible that these two signals are caused by unspecific binding. 

The structure of I19L was determined in aqueous solution at pH 6.8 by NMR [142]. Chemical shift values and the scarcity of cross-peaks in NOESY and ROESY spectra indicated that the peptide is mostly disordered in solution, although it has a helical propensity at residues 10-14 (Fig. 36). The bound conformation was investigated by means of tr-NOESY and STD experiments using a 30 1 excess of peptide relative to tubulin. The presence of intense, negative crosspeaks in the tr-NOESY spectrum proved that there is a fast binding equilibrium (Fig. 36). Unspecific binding was ruled out based on the absence of tr-NOE peaks in a control experiment where the protein was BSA instead of tubulin. The existence... 

Assay miniaturization helps to reduce the consumption of very expensive assay reagents. The problems encountered as one attempts to miniaturize an assay relate to the change in surface-to-vol-ume ratio, lowered sensitivity, and low volume dispensing of materials. For instance, as one moves to smaller volumes, the surfaces available for unspecific binding increase relative to the volume. Furthermore, the smaller the volume, the less the amount of product-sensing material can be added thus the sensitivity of the assay is reduced. 

Irreversible inhibitors have a major advantage over their reversible counterparts in that their action is sustained after systemic clearance. However, the pharmaceutical industry usually does not make use of irreversible inhibition principles, because unspecific binding to proteins other than the target enzyme may lead to considerable toxicity. Since most of the current cancer therapies are unspecific and very toxic, the irreversible kinase inhibition principle may well be applied in this research area, provided that an improved risk/benefit ratio is observed [4e],... 

---