Protein unreactive

For large quantities of protein, unreacted acrylodan can also be removed by passing the protein over a size exclusion column. It should be noted that small traces of free acrylodan do not have a significant effect on assay performance. However, free acrylodan is reactive and this can have unknown consequences when compounds are introduced during screening. 

We ll see in Section 26.7 that this DCC-induced method of amide formation is the key step in the laboratory synthesis of small proteins, or peptides. For instance, when one amino acid with its NH2 rendered unreactive and a second... 

Figure 26.9 X-ray crystal structure of citrate synthase. Part (a) is a space-filling model and part (b) is a ribbon model, which emphasizes the a-helical segments of the protein chain and indicates that the enzyme is dimeric that is, it consists of two identical chains held together by hydrogen bonds and other intermolecular attractions. Part (cl is a close-up of the active site in which oxaloacetate and an unreactive acetyl CoA mimic are bound.

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

Phenylalanine and tryptophan contain aromatic side chains that, like the aliphatic amino acids, are also relatively non-polar and hydrophobic (Figure 1.4). Phenylalanine is unreactive toward common derivatizing reagents, whereas the indolyl ring of tryptophan is quite reactive, if accessible. The presence of tryptophan in a protein contributes more to its total absorption at 275-280nm on a mole-per-mole basis than any other amino acid. The phenylalanine content, however, adds very little to the overall absorbance in this range. 

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

Separate modified protein from unreacted SATA and reaction by-products by dialysis against 50 mM sodium phosphate, pH 7.5, containing ImM EDTA or by gel filtration on a Sephadex G-25 column (Pharmacia) using the same buffer. 

Remove unreacted N-acetyl homocysteine thiolactone and reaction by-products by gel filtration or dialysis against lOmM sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.2. Other buffers suitable for individual protein stability may be used as desired. For the silver nitrate-containing reaction, removal of the silver-thiourea complex may be done by adsorption onto Dowex 50, and the protein subsequently eluted from the resin by 1M thiourea. Removal of the thiourea then may be done by gel filtration or dialysis. 

The relative reactivity of a-haloacetates toward protein functionalities is sulfhydryl > imid azolyl > 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... 

Purify the conjugate from unreacted protein or unreacted dendrimer using gel filtration chromatography with a matrix having an exclusion limit appropriate to accommodate the size of the molecules being separated (i.e., a HiPrep 16/60 column packed with Sephacryl S-200 HR, GE Healthcare). 

Remove unreacted NHS-fluorescein and reaction by-products by gel filtration or dialysis. Continue to protect all labeled protein solutions from light. 

Centrifuge and wash the particles at least 3 times with buffer to remove unreacted protein and ethanolamine. Finally, suspend the particles in a suitable storage buffer containing a preservative. 

Purify the modified protein from unreacted biotinylation reagent and reaction by-products using dialysis or gel filtration. Complete removal of the excess reagent is necessary to provide accurate measurement of the biotin incorporation level by absorptivity. 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

Separate unreacted protein from modified liposomes by gel filtration using a column of Sephadex G-75 or by centrifugal floatation in a polymer gradient (Derksen and Scherphof, 1985). 

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