Extraction streptavidin

Fig. 5.13 On-line continuous-flow, multi-protein biochemical assay. MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. Extracted-ion chromatograms of (a) digoxigenin (m/z 391.5), (b) biotin (m/z 245.0), (c) fluorescein-biotin/streptavidin (m/z 390.0) assay and (d) digoxin/anti-digoxigenin m/z 798.5) assay. Triplicate injections were performed WITH blank (peaks 1-3), 1 pM digoxigenin (peaks 4-6) and 1 pmol biotin (peaks 7-9).

Avidin is a glycoprotein which was initially extracted from chicken egg white and which exhibits four deep binding pockets for biotin. Streptavidin is an analogous protein with a molecular weight of about 70 kD which is prepared from the recombinant bacteria strain Streptomyces avidinii and which... 

Biotin and biotin analogs Infant formula Protein precipitation using concentrated hydrochloric acid neutralization with 6 M NaOH lipid extraction with n-hexane Precolumn Microsorb C18 (15 X 4.6 mm, 5 jam Rainin). Analytical Microsorb C18 (250 X 4.6 mm, 5 /zm Rainin). Isocratic 100 mM phosphate buffer, pH 7.0 + methanol (80 + 20, v/v). 0.4 ml/min. Postcolumn reaction system UV absorbance at 220 nm followed by streptavidin-fluorescein isothiocyanate (2.0 mg/L) knitted open tubular reaction system (10.0 m x 0.5-mm ID) at a flow rate External standardization. 184 Linear range = 0.08-1.00 fjM biotin. LoD = 0.02 /zM or 97 pg biotin at SNR = 3. Repeatability CV 3.5% for biotin in infant formula. 

In addition to the attempts at unraveling the modification site, Dive and coworkers also constructed two biotinylated A/BPs, 22 and 23 (Scheme 3c), and used these to study the difference in affinity- and photoaffinity MMP enrichment from a complex proteome [57]. For this, tumor extracts were spiked with hMMP-12 and hMMP-8, after which compounds 22 and 23 were applied, followed by streptavidin-coated magnetic beads for MMP pull-down. Affinity-based labeling with 23 appeared superior to photoaffinity-based labeling with 22 in terms of quantity of captured MMPs and identification of the tryptic fragments by mass spectrometry. 

The same authors also examined the S-nitrosylation of proteins by nNOS, in neuronal tissue from mice (Figure 11.10). Conversion of nitrosylated to biotinylated cysteines was done as above. Biotinylation was, in this case, used to extract the (formerly) nitrosylated proteins from the total mixture of cellular proteins by selective binding to solid-phase attached streptavidin. After retrieving the bound material by reduction with excess free thiol, the samples were run on a gel, blotted, and individual proteins detected using an-... 

Why did the authors not use the anti - S-N=0 antibody instead Maybe they didn t think of it. However, I suspect that the antibody might not possess sufficient affinity to allow for stringent washing of blots and columns. The biotin-streptavidin interaction is very stable and thus withstands the stringent washing steps required for selective detection within complex protein mixtures such as whole cell extracts. 

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