Acid soluble autoradiography

Usually autoradiography is used to detect high molecular weight compounds which are acid or ethanol insoluble, e.g. DNA. These have been made radioactive by incorporation of a low molecular weight, acid soluble precursor and the fixation process must ensure that excess precursor is removed. 

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 ... 

Because cell-free methods are highly efficient in their incorporation of amino acids, only minute quantities of radiolabeled amino acids are required for analysis by autoradiography. Background translation of endogenous mRNA is also extremely low. This is advantageous because expression conditions can be rapidly screened for their effect on aggregation, solubility, and proteolytic susceptibihty of the expressed construct. Low background translation also means that total and soluble protein yields can be analyzed by auto-... 

Moeton and Rogees [49] have been able to distinguish various relabelled soluble ribonucleic acids through TLC on PEI-cellulose and other ion exchangers. Their solvents were the upper phase of the system isopropanol-formamide-phosphate buffer, pH 6.2 (20 + 5 + 60) [32] or the lower phase of the mixture n-butanol-water-tri-n-butylamine-acetic acid-di-n-butyl ether (100 + 130 + 10 + 2.5 + 29) [107] the fractions were detected by autoradiography [49]. 

---