Double-label dilution experiment

In the double-label dilution experiment described here, the specificity of a particular metabolic pathway was examined by monitoring the fate of an existing labeled chemical bond In a molecule entering that pathway. Naturally, the complementary experiment can also be performed in which the formation of a labeled chemical bond from two separate specific labels Is observed directly using double-cross polarization nmr. 

The fraction, f, of double label in the cultured cotyledons is, of course, not known. This number can be determined by a plot of AS/Sq against (l-e /T ) which is a straight line with slope f. Since virtually all of the labeled carbon in these cotyledons is known to be carbonyl (10), an appropriate T is 3.64 msec. Values for f for sister cotyledons in a doublelabel dilution experiment are shown in Table I, and are reported as percent amide (10). 

Primary antibodies. Affinity-purified primary antibodies are diluted in PBS/BSA (bovine serum albumin). The dilutions must be adjusted for each individual primary antibody. Typical dilutions for rabbit polyclonal antibodies range between 1 50 and 1 500. For double-labeling experiments, use primary antibodies raised in two different species. 

Method 3 Fluorescence-conjugated secondary antibody for double-labeling experiments, use fluorescence-conjugated secondary antibodies raised to the corresponding type of primary antibody the dilution of each antibody needs to be determined individually. 

With very fast growing tissues such experiments are hard to perform because the data must be corrected for both the increase in cell number and in total DNA content which dilutes radioactively labeled DNA by newly synthesized DNA molecules. Therefore, the error becomes very large if the cell number of a given tissue doubles in less than a week. 

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