Radiolabelled protocols

The best radiolabeling technique for SASD is to use the Iodogen method (Shephard et al., 1988) described in Chapter 12, Section 3. The following suggested protocol for using SASD was based on the method described in the Thermo Fisher Catalog. 

Each bead can iodinate up to 500 pg of tyrosine-containing protein or peptide. This translates into an oxidative capacity of about 0.55 pmol per bead. The rate of reaction can be controlled by changing the number of beads that are used and altering the sodium iodide concentration added to the reaction. Reaction volumes of 100-1,000 pi are possible per bead. The following protocol is suggested for iodinating proteins. Optimization should be done to determine the best incorporation level to obtain good radiolabel incorporation with retention of protein activity. 

FIGURE 15.8. Mouse local lymph node assay (LLNA) (ICVAM protocol). Modification using flow cytometry instead of radiolabeling is preferable. 

One method for the labeling of liposomes with chelator, hexamethylpropyleneamine oxime (HMPAO) was developed by Phillips et al. (16). Lipophilic HMPAO enters the liposome where it interacts with glutathione and becomes converted to the hydrophilic form, which is trapped in the liposome. A detailed protocol for radiolabeling liposomes using Tc-HMPAO has been reported (3). In a typical experiment, 10 to 15mCi (370-555 MBq) of Tc in the form of sodium pertechnetate... 

In addition to ARSAC approval, the protocol must also be approved by ethics committees in the normal manner for studies in man. The study should be conducted in between four and eight consenting subjects, in facilities where any spills of radiolabelled materials can be contained and monitored. Normally, subjects will be required to provide blood samples and to collect all excreta for a period determined by the known or estimated half-lives of the parent compound and metabolite. With cooperative subjects, recoveries of radioactivity should be close to 100%. Samples will be assayed for radioactivity and by cold chromatographic methods, and every attempt should be made to identify major metabolites... 

Traditionally, HAT activity is measured with a discontinuous radioactive filterbinding assay, which uses pH]acetyl-CoA as a histone acetyltransferase substrate [46]. The transfer of [ H]acetyl-groups to the histone substrate by histone acetyltransferases is detected by liquid scintillation counting of pHjacetylated histones, which are retained on a phosphocellulose disk. Due to its discontinuous character, this assay is technically problematic and not ideal for kinetic analysis. Hence, other assays that work with radiolabeled acetyl-CoA have been described that are suitable for a higher throughput. These work with streptavidin-covered beads [47] or a variant of the SPA with microtiter plates that contain a scintillant (FlashPlates) [48]. But as all these protocols are based on radioactively labeled substrates, they apparently show the same disadvantages that were described for the radioactive HDAC assay protocols. Therefore, nonradioactive assays have been developed to study histone acetyltransferase activity. 

The following sections describe the principal reagents available for producing tagged molecules using fluorescent probes, radiolabels, and biotinylation techniques. In many cases, suggested protocols are included (or other sections referenced) for the use of these probes. 

Comparative assessments of purification protocols are generally difficult, and wide ranges of activities have been reported. ACV synthetases have been assayed by either employing a peptide adsorption resin (Porapak) [29,48] or high performance liquid chromatography (HPLC) detection of the derivatized tripeptide [26,27], The adsorption assay, employing radiolabeled amino acids, has been... 

Optimized protocols for radiolabeling scVEGF with "mTc for SPECT imaging and 64Cu for PET imaging are described in Subheadings 3.5.-3.7. and 3.8., respectively. 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

In the usual experimental protocols involving PAF, the objective often is to determine whether PAF is formed under stimulatory conditions or whether it has been metabolized by cells, cellular extracts, or specific enzymes. In most instances, the reactions can be followed only through the use of radiolabeled precursors and certain simple analytical procedures. The methodology to be... 

The protocol can be adapted to measure receptor recycling. If radiolabeled antibodies are internalized into cells by incubation at 37°C in the presence of chemokine or other agents, the acid-elution procedure can be used to remove radiolabeled antibodies remaining on the cell surface. If the acid-elution medium is not reduced below pH 3.0, and the washes kept brief and performed at 4°C, then the cells can be returned to 37°C culture (care should be taken to ensure that the endocytic-trafficking properties of cells are not perturbed by the low pH treatment). During a subsequent incubation at 37°C, receptors that recycle will return antibodies to the cell surface. These antibody molecules can be assessed by measuring the radioactivity that becomes accessible to a second round of acid elution. 

Fibroblasts were selected because they are readily cultivated and radio-labeled and are available from skin explants of a variety of species. Autoradiograms of radiolabeled cellular proteins are more diverse and easily analyzable for molecular systematics than alternatives such as silver-stained serum or erythrocyte protein patterns. We have achieved nearly 100% success rates at minimal discomfort and risk to human subjects by establishing cultures from 3 mm punch biopsies from the upper buttock. Local lidocaine anesthesia is used. Samples are collected following informed consent and under an approved human research protocol. Other species are sampled by dart gun8 or while sedated. Skin samples can be collected from various body sites without compromising the 2D electrophoresis metric, which does not rely on quantitative differences in protein expression. To comply with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), tissues collected from wild and captive exotic animals must be obtained under specific permits issued by the U.S. Fish and Wildlife Service. 

As an alternative to the use of most, if not all, of the procedures described in the preceding section can be done using antibodies conjugated to an enzyme that converts a substrate into a colored or fluorescent product see also this vol.. Chapter 10) that is either soluble (plate assays) or insoluble (Western blots) (sec Note 11). The protocols for the use of either radiolabeled or enzyme-conjugated antibodies are the same until the completion of... 

---