Biotin unreacted

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. 

However, a more favourable pathway is used, employing a more reactive nucleophile. Rather than using the enolate anion derived from acetyl-CoA, nature uses the enolate anion derived from malonyl-CoA. Malonyl-CoA is obtained from acetyl-CoA by means of an enzymic carboxylation reaction, incorporating CO2 (usually from the soluble form bicarbonate). Now CO2 is a particularly unreactive material, so this reaction requires the input of energy (from ATP) and the presence of a suitable coenzyme, biotin, as the carrier of CO2 (see Section 15.9). The... 

Carbon dioxide is a normally unreactive material, and combination with biotin requires the input of energy (from ATP). Carbon dioxide is usually present... 

Either of the foregoing mechanisms requires that the ureido anion of biotin attack the rather unreactive carbon atom of carboxyphosphate. Another alternative, which is analogous to that suggested for PEP carboxylase (Eq. 13-53) is for carboxyphosphate to eliminate inorganic phosphate to give the more electrophilic C02 (Eq. 14-9, step a). The very basic inorganic phosphate trianion P043 that is eliminated could remove the proton from Nl of biotin to create the biotin ureido anion (step b), which could then add to C02 (step c).22... 

Isotype-matched unconjugated, biotin-conjugated, or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers... 

Remove unreacted biotin molecules by dialysis against PBS. 

Dilute the reaction mixture by adding 4 volumes of H20. Remove unreacted BMCC-biotin through a 30kDaMWCO filter (Millipore) by centrifugation for 8 min at 11 000 x g. Repeat this step to ensure that all unreacted BMCC has been removed before proceeding to the partitioning step (Section 8.3.6). 

The unreacted NHS-biotin has to be removed from the enzyme solution by ultrafiltration with 0.1 M phosphate buffer (pH 7) at 6 C. 

The final reaction, catalyzed by biotin synthase, involves the insertion of sulfur between the unreactive methyl and methylene carbons of dethiobiotin. The enzyme has an iron-sulfur box, and requires NADPff and a ferredoxin or flavo-doxin reducing system. S-Adenosylmethionine is also required, and is cleaved to yield methionine and a 5 -deoxyadenosyl radical during the reaction. Biotin synthase is a member of the radical SAM family of enzymes, in which the catalytic 5 -deoxyadenosyl radical is formed from S-adenosylmethionine,... 

Methodologies to produce reactive SAMs, such as inter-chain anhydrides, have been adapted to forming patterns of SAMs with pCP (45-47). Monolithic films of SAMs containing reactive pentafluorobenzyl esters were converted into patterned amides by using a stamp containing a primary amine. In the areas of contact, the amine reacts with the activated surface to form a patterned amide. Printing of benzenesul-fonamide was detected by binding cabonic anhydrase (CA) to the sulfonamide-derivatized SAM. Benzenesul-fonamide binds to CA with a dissociation constant of less than 10 M. Similarly, the stamp can be treated with poly(ethylene imine) (PEI) to transfer a pattern of amide-coupled PEI, where the pendant, unreacted amines of PEI can be used for subsequent nucleophilic reactions. This procedure can introduce patterned biotin... 

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