Blocking reagents

The utility of alkenylcarbene complexes as C3 building blocks in the [3C+2S] cycloaddition reaction has been demonstrated by the wide variety of five-membered hetero- and carbocycles obtained when these complexes are treated with several C2 building block reagents. This impressive chemistry will be briefly discussed in the next few sections. 

Sulfonated or sulfomethylated lignins are reacted with phenol-blocking reagents, such as ethylene oxide, propylene oxide, or butylene oxide [1571]. 

The choice and application of a specific blocking reagent can produce a modified macromolecule with unique and useful properties. Many of the common blocking reagents are discussed... 

Habeeb, A.F.S.A., and Atassi, M.Z. (1970) Enzymatic and immunochemical properties of lysozyme. Evaluation of several amino group reversible blocking reagents. Biochemistry 9, 4959—4944. 

Some patients will have repeatedly nonspecific reactivity in an ELISA assay, since the HIV-1 antigen used for the antibody assays is produced in cultured human T cells. It is not unexpected that occasional false positive assays occur in human sera from individuals with autoimmune diseases a history of multiple pregnancies or multiple transfusions or antibodies to certain class II histocompatibility antigens (especially HLA-DR4). Block reagents have been added to specimen diluents to minimize cross-reactions in these sera. This necessitates the use of confirmatory tests, especially the Western blot. With the use of both ELISA and Western blots, false positives decrease to less than 1 per 100,000. 

Combinatorial chemistry is a laboratory chemistry technique to synthesize a diverse range of compounds through methodical combinations of building block components. These building blocks (reagents) are added to reaction vessels, and the reactions proceed simultaneously to generate an almost infi-... 

Before starting experiments with human or animal tissue samples, it is extremely important to optimize in vitro experimental conditions. With a purified template nucleic acid, standardize RT and PCR conditions. Check the specificity and crossreactivity of primers and probes. Sometimes it is necessary to alter MgCl2 concentration under in situ reaction conditions. The blocking reagent for filter hybridization could be different than the in situ protocol. (I use 1 % purified casein solution for filter hybridization and 3% BSA for in situ signal detection.)... 

This enzyme [EC 3.4.16.6], also known as carboxypeptidase KEXl and carboxypeptidase SI, is a member of the peptidase family SIO and catalyzes the hydrolysis of a peptide bond located at the C-terminus of a polypeptide with a preference for a C-terminal arginine or lysine residue. This broadly specific enzyme is inhibited by the diisopropyl fluorophosphate and thiol-blocking reagents. The pH optimum ranges from 4.5 to 6.0. 

This enzyme [EC 3.4.19.3], a member of the C15 peptidase family, is also known as pyroglutamyl-peptidase 1,5-oxoprolyl-peptidase, pyrrolidone-carboxylate peptidase, and pyroglutamyl aminopeptidase. This hydrolase catalyzes the conversion of a 5-oxoprolyl-peptide to produce 5-oxoproline and a peptide. The enzyme will not act on the 5-oxoprolyl peptide if the adjacent amino acid is l-proline. Enzyme activity is inhibited by thiol-blocking reagents. 

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

The following protocol is an example for blocking, working very well in numerous cases, but optimization by use of other blocking reagents is worth checking every time. 

B 3. An economical way to block a blotted membrane is to incubate it in a 10% solution of nonfat milk powder. How does this solution function as a blocking reagent ... 

The relative reactivity of a-haloacetates toward protein functionalities is sulfhydryl > imidazolyl > thioether > amine. Among halo derivatives the relative reactivity is I > Br > Cl > F, with fluorine being almost unreactive. The a-haloacetamides have the same trend of relative reactivities, but will obviously not create a carboxylate functional group. The acetamide derivatives typically are used only as blocking reagents. 

ELISA of sheep IgM (antigen) was conducted in a PDMS chip. The capture antibody (rabbit anti-sheep IgM-HRP) was added. Then the fluorogenic substrate (HPPA) was added for fluorescent detection. The conventional blocking reagents (0.5% w/v BSA, 0.5% w/v casein, 0.5% v/v Tween-20), which normally worked for the polystyrene ELISA plate, did not work with the hydrophobic PDMS surface. To solve this problem, 4% PEG and 7% normal rabbit serum were included in the above solution for effective blocking [173]. 

Several reversible blocking reagents for amino groups in proteins have been reported (see Table I). Succinyl derivatives cannot be removed easily from succinylated proteins under mild conditions however, Braunitzer et al. (43) have shown that tetrafluorosuccinyl derivatives could be removed at pH 9.5 and 0°C (see Reaction 1) ... 

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