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Use the channel ratios of the standards to calculate the amount of each isotope in each channel. 416)(800) = 333 cpm 3 H in channel A = 500 - 333 = 167 Total 3H = 167 cpm Total 14C = 800 + 333 = 1133 cpm *In other words, at these channel settings there is no significant amount of 3H in channel B and all of the radioactivity can be assumed to be 14C. 45 46 CHAPTER 5--pH AND BUFFERS Maintaining the proper pH during experimental procedures is often one of the most critical factors in determining whether an experiment will work.

Many proteins bind to ligands or other substances and this binding activity is measured. , stimulation of cell division). In cases where the protein of interest has no measurable activity or the activity is unknown it may be possible to generate antibodies against the protein and develop an immunoassay (to be discussed later). If antibodies against such a protein are not available, the assay may simply be the amount of a protein band on a Commassie blue-stained gel following electrophoresis (to be discussed later).

Autoradiography of gels allows the 2. Cover with a photographic emulsion. identification and quantitation of specific proteins or 3. Expose and develop. nucleic acids (discussed later). It is also possible to 4. Examine under microscope. localize radioactive markers to particular cells in tissue sections or subcellular structures within cells by autoradiography. The typical procedure is to cover cells with a photographic emulsion (Box) and develop the emulsion after sufficient exposure. Dark grains (or spots) will appear over the cells or structures which contain the radioactive marker.

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