Double-label dilution

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. 

With a 13C label at the methide center, the presence of reactive methide intermediate can be verified and complex reaction products can be inventoried and eventually identified. The only limitations are the synthesis and cost involved in incorporation of the 13C label. As a rule we, only use 13C-labeled dimethylformamide and NaCN as starting materials because of their low cost and availability. Another limitation of enriched 13C-NMR monitoring is dilution of the enriched label to natural abundance levels. Currently, we are developing isotope-editing techniques that utilize unnatural 13C double labels to solve this problem. 

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). 

By diluting the double-labelled PBG with four parts of unlabelled PBG, and then incubating the mixture with deaminase and cosynthetase (and other enzymes that take the labelled uro gen III through to protoporphyrin IX labelled in exactly the same carbon atoms), most of the uro gen III... 

Therefore, in the absence of glucose-6-phosphatase as found in G.S.D.I it is expected that this decrease will not be present since double-labelled glucose present in the circulation will not be diluted with glucose liberated from the liver after having lost while passing through the isomerase reaction. 

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. 

Note For double labeling, treat embryos with the first primary antibody, then perform steps ll-12a (i-ii) or steps ll-12b (i-ii). Rinse five to six times in PBT. Incubate in second primary antibody (appropriately diluted) at 4 C overnight. Proceed with steps 11-12. 

Lifson and coworkers [48] used isotope dilution techniques in form of the double-labelled water method to evaluate the CO2 production and the origin of the o.xygen expired in CO2 In the carbon dioxide entry technique CO2 production is determined and transformed to energy units by means of the RQ value [49]. Both techniques were applied to larger animals, but they are not applicable to short time investigations. 

Remove excess PBS and immediately apply 50 pi double-FAM LNA (see Note 2) probe solution and gently shield with cover glass (see Note 3). The probe solution is prepared as follows denature LNA probe and dilute the probe in Exiqon ISH buffer. For example, for 2 ml hybridization mix containing 20 nM double-FAM-labeled miR-21 LNA probe (from 25 pM probe stock), transfer 4 pi into the bottom of a 2 ml nonstick RNase-free tube and place the tube at 90°C for 4 min. Spin down shortly using a tabletop centrifuge, and immediately... 

For quantitative evaluation a set of 6 serial dilutions of the original sample that should have an accurately known concentration of -2.0%. The concentration can be adjusted to give the optimum odor data. Each sample is diluted by a factor of 3 from the previous sample (i.e., 1 ml sample diluted with 2 ml of solvent) and then given a random number using a double blind labeling method. The samples are sniffed in random order. 

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