Protein double labelling

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

Kainosho M, Tsuji T, Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution, Biochemistry, 21 6273-6279, 1982. 

It should be emphasized that the foregoing experiments were not designed to study the mechanisms of heat-induced protein interactions in milk. They merely serve to illustrate the utility of radiolabeled milk proteins in studying this very complex phenomenon. The radiolabeling of other milk proteins along with double-labeling experiments should prove very instructive in future research on the heat stability of milk. 

In general, all 18 bic nic L-amino acids may be uswd for labeling proteins. They may be combined for so-called double labeling, and even triple labeling is sometimes... 

Double-labeled proteins from rat liver cytosol ( C in long-lived, in short-liv J proteins after in vivo labeling) have been used as substrates for proteinases in vitro. The differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases in the presence or absence of different effectors enabled conclusions to be drawn concerning their role in in vivo turnover. The main activity of lysosomal proteinases at pH values of 6.1 and 6.9 was found to be caused by thiol proteinases which decompose short-liv l cytosol proteins preferentially. Autolysis of double-labeled cell fractions showed a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes >. 

The method of double labeling has been appli in other studies of protein turnover. Jung > has used the radionuclides and C. A similar approach was chosen by Konno et al. to investigate the relative turnover rate of soluble proteins in rat liver. 

Apart from the wide ran of staining techniques, fluore nt and radioactive labels are often used. FITC is used for protein detection following SDS-electrophoresis, e.g. Rat hemoglobin fractions have been assayed by labeling with Double labeling with and makes it possible to detect proteins from two different... 

In order to pursue heteronuclear multidimensional NMR experiments, a bacterial system for expression of apoLp-III has been developed which allows facile production of 150 - 200 mg/L l N-iabeled apoLp-III or 100 - 125 mg/L 15N/l3c-double labeled apoLp-III. Figure 2, panel A shows the IH- n HSQC spectrum of a 1.0 mM solution of lipid-free, uniformly N-labeled apoLp-III. Panel A also indicates that, although the chemical shift dispersion in the H-dimension is rather small (6.5 ppm to 9.5 ppm), it is generally upfield shifted, consistent with the fact that the protein secondary structure is predominantly a-helix (13). The chemical shifts in the N-dimension are well-dispersed which results in good separation of the overall crosspeaks. However, certain regions in the spectrum are still crowded as shown in Figure 2. 

For proteins smaller than 10 kDa, the 15N-only approach may prove adequate. If not, double labeling with both 15N and 13C will be necessary, and assignment will be most readily achieved using triple resonance... 

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