Screening by Flow Cytometry

Library Screening by Flow Cytometry A. Ligand Binding 

Flow Cytometric Analysis of the Effect of the Rate of Random Mutations 

A detailed analysis of the effect of the mutational load on the distribution of protein activity within a mutant population has been carried out 

In other studies, Wain-Hobson and co-workers have selected active variants of dihydrofolate reductase (DHFR, 78 a.a.) from hypermutated libraries (Martinez et al., 1996). Amino acid substitutions were found in all but six residues. Three rounds of mutation and selection led to the isolation of DHFR mutants in which 22% of the amino acids had been substituted. Finally, Palzkill and co-workers have carried out a very systematic mutagenesis of nearly all codons in /1-lactamase (Huang et al., 1996). In this case 43 out of 263 residues were found to not tolerate amino acid substitutions. For DHFR and /8-lactamase, the two enzymes that have been subjected to systematic mutagenesis of nearly every residue, the fraction of invariable residues was roughly of the same order (9% and 16%, respectively). 

One mechanism that may be important in determining protein plasticity is the occurrence of second site suppressor mutations that serve to compensate for deleterious amino acid substitutions that interfere with 

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MAbs produced by immunization with peptide may be screened first by ELISA to identify peptide specific antibodies. However, not all peptide specific antibodies will recognize the cell surface-expressed receptor. MAbs reactive with peptide must be subsequently screened by flow cytometry on receptor transfectants, cell lines, or leukocytes known to express the receptor in order to identify those which recognize native receptor. Alternatively, the fusion can be screened directly by flow cytometry to identify antibodies which recognize native receptor (see Subheading 3.3.2.),... 

MAbs produced by immunizing with receptor transfectants must be screened by flow cytometry—first against receptor transfectants to select for immunore-active MAbs to the immunogen and then against the parental cell line to eliminate those immunoreactive antibodies recognizing determinants other than the transfected receptor. Mice will make antibodies to cell surface determinants other than the receptor of interest even when a syngeneic mouse cell line is used as the parental line for transfectants. 

The screening of protein libraries by flow cytometry offers the following advantages ... 

Screening for Cell Surface Binding by Flow Cytometry... 

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

Visual screening, facilitated by the color of the alkaloid, yielded highly productive cell lines of berberine (2/). The strong fluorescent properties of serpentine allowed the determination of its concentration in individual cells by flow cytometry, and subsequent sorting of the cells with high contents yielded a highly productive cell line (23). The same technique was used for berberine-containing cells (24). 

FD-NC maintenance. The isolation of FD-NC precursors was managed by flow cytometry. Then, FD-NC was cultured and amplified in the presence of FGF2 and EGF for 2 weeks, which provides large batches ready for subsequent screening. 

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