By Henry Frederick Baker
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Extra info for An introduction to the theory of multiply periodic functions
8. Faye and Chrispeels (1985). 9. Gorelick et al. (1983). 10. Johnson et al. (1984). 1 1 . Lin and Kasamatsu (1983). 12. Mandrel1 and Zollinger (1984). 13. Miskimins et al. (1985). 14. Ramirez et al. (1983). 15. Saravis (1984). , 1982). An added advantage to the exclusive use of a detergent is that after probing, the blot can still be stained for protein. X. PROBING AND WASHING The quenched blot is now ready to be probed. ). A rule of thumb is that one should probe in a quenching solution. The rationale behind this is to prevent nonspecific adsorption of the probe to irrelevant areas of the blot.
A list of quenchers is given in Table 111. An alternative approach to quenching with proteins is the use of detergents. Conceptually, detergents are not really quenchers because they do not sequester binding sites but rather reduce the nonspecific “stickiness” of the probe and filter. , Triton X-100, Nonidet-P 40,and Tween 20)have been used. The use of detergents is attractive because these substances are cheap, readily available, easily handled, and rather efficient. Their major drawback is that they definitely interfere with the association of protein with the immobilizing matrix.
Iii) Negative staining of the blot is also possible. This is achieved by incubating the blot in a dilute solution of alkaline phosphatase (10 pg/ml). The blot is then rinsed in PBS and reacted for enzyme activity as described above. XV. TROUBLESHOOTING In a multistep process such as immunoblotting there are numerous points that may be problematic. In this section, I deal with three problem areas and provide possible explanations that sometimes help in overcoming them. 48 JONATHAN M. GERSHONI 1.