Nonspecific binding

One ml of lysis buffer should be added to 10 /il/IP of protein G-Sepharose. The tube should be flicked to mix the contents and then centrifuged at 8000 rpm for 1 min. The supernatant is removed, 500 pi of lysis buffer, 1 to 2 /ig/IP of the relevant antibody are added, and the tube is rotated at 4° for 1 h. The beads are collected after a quick spin and washed twice with 1 ml of lysis buffer. Total cell lysate is added to the beads and the final volume increased to 500 pi with lysis buffer. The tube is rotated at 4° for 1 to 2 h, and the beads are washed twice with 1 ml of lysis buffer containing 0.5 M NaCl before further analysis. To reduce any nonspecific binding, the total lysate can be preabsorbed with protein G Sepharose for 1 h at 4° in order to remove components that bind nonspecifically to the beads. Alternatively, the extract may be incubated with the specific antibody for 1 h at 4°, and the... 

Remove excess crosslinker and reaction by-products by dialysis or size exclusion chromatography. For small quantities of bait proteins, dialysis may be the better choice, because gel filtration columns often bind nonspecifically enough protein to make recoveries unacceptably low. 

Once an agent is topically applied in the oral cavity, the free drug can act at the primary site (i.e., bacteria in the plaque), or it can be partitioned to compartments where the drug binds nonspecifically. These drug reservoirs include the enamel, dentin, and/or cementum of the tooth, the oral mucosa, the organic and inorganic components of plaque, and salivary proteins. 

Is nitrocellulose antigenic Some workers have been unable to achieve good results by immunization with nitrocellulose-bound protein. They reproducibly obtain antisera directed against nitrocellulose We found that in every case, this resulted from injecting powdered nitrocellulose m Freund s adjuvant using adjuvant actually increases the production of low affinity IgM that binds nonspecifically to nitrocellulose. We have never observed this effect in our experiments when the technique described here was followed strictly... 

By way of a completely different type of functional analysis, we can look at the use of tracking dyes to label and follow cells. Cells can be stained with lipophilic fluorochromes or with fluorochromes that bind nonspecifically to all proteins. An example of the lipophilic fluorochromes are the so-called PKH dyes that insert themselves into the bilayer of cell membranes. The dye carboxyfluorescein succinimidyl ester (CFSE) binds to the free-amino groups on all cell proteins. These two types of dyes can be used to stain cells with considerable stability. The stained cells can then be injected into an animal and tracked to their homing location. 

Specific binding = Total binding - Nonspecific binding... 

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