Autoradiography of gels

There are two problems with autoradiography of gels (Laskey 1980). 

Fio. 2. Plasmin and uPA-catalyzed cleavage of suPAR(I-III). (A) 35S-labeled recombinant suPAR(I-III) was purified on an immunoaffinity column with the domain I-specific mAb R3 immobilized [86], Lane 1 is 10 nM suPAR(I-III), lane 2 is 10 nM suPAR(I-III) incubated for 2 hours at 37 °C with 50 nM plasmin (pli), lane 3 is 10-nM suPAR(I-III) incubated for 20 hours at 37°C with 500-nM uPA, and lane 4 is 35S-labeled domain I purified from cell culture media on an immunoaffinity column with the domain I-specific mAb R9 immobilized, after removing suPAR (I—III) from the media by immunoaffinity chromatography, employing the domain Ill-specific mAb R2. (B) Ten nM suPAR(I-III) was preincubated with 67 nM of the indicated mAbs for 2 hours at 37°C prior to addition of either 50 nM plasmin (lanes 2,3,4, and 6) or 500 nM uPA (lane 5) and continued incubation for 2 or 20 hours at 37 °C. Lane 1 is suPAR(I-III) alone, lane 2 is suPAR(I-III) incubated with plasmin only, lane 3 shows preincubation with a subtype control mAb, lanes 4 and 5 show preincubation with mAb R3, lane 6 shows preincubation with the domain Ill-specific mAb R4. The radioactive samples have been separated by SDS-PAGE prior to autoradiography of the dried gel. 

C. C. Chery, E. Dumont, R. Comelis, L. Moens, Two-dimensional gel electrophoresis of selenized yeast and autoradiography of 75Se-containing proteins, Fresenius. J. Anal. Chem., 371 (2001), 775-781. 

Inasmuch as ATP and polyP have similar phosphorylating potentials, the ability of [32P] (polyP) to phosphorylate the CaATPase was examined by autoradiography of an SDS-PAGE gel (Figure 21 A). 

Top Autoradiography of the 2D gel with pH 3-10 after application of 400 pg protein lysate... 

Fig. 8.2. Device for preparing flat longitudinal slices of cylindrical gels for autoradiography. The block (lower left) is made out of Perspex. The clearance between the walls and the spacers, and between the spacers themselves (expanded drawing at right) is sufficient to allow the framed taut steel wires (upper left) to be drawn through. This provides two flat sections of gel (Fairbanks et al. 1965).

For gel purification, 20CM-00 pmol of 32P-labeled RNA crude mixture is mixed as a tracer with 40,000 pmol of unlabeled crude substrate. Measure the radioactivity of a small aliquot of the mixture in a scintillation counter to calculate the specific activity of the RNA. The dsRNA and ssRNA in the crude mix are separated by electrophoresis in a preparative 8% polyacrylamide gel. After visualization by autoradiography, the region of the acrylamide gel containing the dsRNA is excised with a razor blade and the RNA is extracted from the gel. After gel purification, the dsRNA substrate is suspended in buffer I, and the radioactivity of an aliquot is measured on the scintillation counter. From the specific activity of the dsRNA, the amount of gel-extracted substrate can be determined. This is called the cold substrate because the specific activity is lower than that of the hot substrate, which is freshly labeled with 33P for use in the assay. Since a small amount of the purified dsRNA is labeled with 32P, aliquots of gel-purified cold substrate should be stored in an acrylic (3-radiation storage container until they the radiation has decayed with time. 

Rapid sequencing techniques have been developed to further the analysis of DNA molecules. DNA can be sequenced by controlled interruption of replication. The fragments produced are separated by gel electrophoresis and visualized by autoradiography of a label at the 5 end or by fluorescent tags. 

Table 1.3. Autoradiography/fluorography of gels (following Laskey 1980). ...

Silver-stained polyacrylamide gels " " have been used in quantitative and fast separations of individual proteins labelled with weak -emitters H) by polyacrylamide gel electrophoresis. The amount of radioactivity in a given band can be determined rapidly by liquid scintillation counting, which was found to be independent of the silver deposition and total protein content, but hnear with respect to the amount of radioactivity in the given band, thus avoiding the lengthy exposures needed, for instance, in the autoradiography of [ H]methionine-labelled proteins separated by polyacrylamide gel electrophoresis. The method was used to determine [ H]mannose and [ H]fucose incorporated into a cell adhesion protein . 

P]NAD is employed as substrate in this reaction, the ADP-ribosylated protein may be identified by the method of polyacrylamide gel electrophoresis followed by autoradiography of the gel. This method was employed in an examination of the membrane proteins of control and desensitized platelets. Coomassie-blue staining of the gels revealed no differences following desensitization, but there was a > 80% loss of the 45 000 Da protein as revealed by [ P]ADP-ribosylation. The autoradiograph and scans are shown in Figure 8.26. 

Fluorescence is used for a wide variety of biomedical purposes. Fluorescence imaging of gels is used to detect DNA fragments following electrophoretic separation. The newer DNA stains provide hi detection sensitivity and can mostly replace the use of and autoradiography. The... 

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