Positive control protein

Christman, M.F., Morgan,R.W., Jacobson, F.S., Ames, B. (1985). Positive control of a regulon for defenses against oxidative stress and some heat shock proteins in Salmonella typhimurium. Cell 41, 753-762. 

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

If a monoclonal antibody was generated by immunization with a full-length native protein rather than a peptide, then the immunized mouse will generate antibodies that recognize both linear and conformationally dependent epitopes. Only a small subset of these monoclonal antibodies will likely be useful for clinical use on formalin-fixed, paraffin-embedded tissue (FFPE) samples. Those that are useful tend to have epitopes that are linear the epitopes are not dependent on the protein s three-dimensional conformation (see Chapter 16). Therefore, for antibodies generated in response to immunization with full-length proteins, the peptides that serve as positive controls will be linear stretches of amino acids derived from the native protein sequence, as listed in protein databases. 

Figure 8.1 The results of IHC of two experiments using Dynabeads (Dynal, New York, NY) coated with biotinylated anti-mouse IgG (first experiment) and protein S-100 (second experiment), (a) Positive control showing red color (S-100) localized in the melanoma cells, (b) Strong positive red color circles all beads coated with biotinylated anti-mouse antibody after the heating AR treatment (first experiment), (c) Using the heating AR treatment, S-100-coated polymer beads show positive red color around the beads as circles (second experiment), (d) Negative control of the first experiment. No red color could be seen for polymer beads (arrows) that had been treated with exactly the same protocol as that of slide (b), but omitting the avidin-biotin-peroxidase (label). Bar = 50pm. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2005 53 1167-1170. See color insert.

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

In a study which examined the role of the protein 2. -globulin in 1,4-dichlorobenzene-induced nephrotoxicity in male rats, NCI-Black-Reiter (NBR) rats, known not to S5mthesize the hepatic form of the 2. -globulin, were administered 500 mg/kg/day 1,4-dichlorobenzene by gavage in com oil for 4 consecutive days. Positive controls consisted of Fischer 344 male rats treated with lindane the results were also compared with those obtained in a group of female Fischer 344 rats treated with lindane. End points examined consisted of kidney lesions and protein droplet evaluation. 2.-Globulin was detected in kidney sections from male Fischer 344 rats, but not in male NBR or female Fischer 344 rats. No lesions or hyaline droplets were detected in treated or control male NBR and female Fischer 344 rats (Dietrich and Swenberg 1991). 

The well-investigated lactose operon of the bacterium Escherichial coli can be used here as an example of transcriptional control. The lac operon is a DNA sequence that is simultaneously subject to negative and positive control. The operon contains the structural genes for three proteins that are required for the utilization of lactose (one transporter and two enzymes), as well as control elements that serve to regulate the operon. 

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