Fluorography

Fig. 1. SDS gel analysis of proteins synthesised by excised maize roots incubated at continuous 40 °C. Roots of 3-day-old maize seedlings were excised and incubated at 40 °C for increasing times as indicated. Labelling with [ Sjmethionine was carried out in the final 20 min of the incubation. Proteins were visualised by fluorography. Mol wt distribution in kDa indicated at left. From Cooper Ho (1983).

Fig. 2. NAPI facilitates H2A, H2B release from nucleosomes that are on positively coiled DNA (A) but not negatively coiled DNA (B). The positively coiled DNA (6.0 kb) with a superhelical density of + 0.05 and negatively coiled DNA (6.0 kb) with a superhelical density of -0.05 were reconstituted with lysine, arginine-labeled histones H3, H4, H2A, H2B by NaCl dialysis from 2.0 M to 1.2 M to 0.6 M to 0.1 M NaCl over a 14 h period. The samples were incubated with NAPI at 35 °C for 5 min and applied to a 5-20% sucrose/100 mM NaCl/40 mM Tris, pH 7.8 gradient. After sedimentation at 200,000 X g for 5 h, fractions were collected and the distribution of DNA (bottom panel) was determined on agarose gel and the distribution of protein (top panel) on SDS-PAGE followed by fluorography. These data are unpublished observations (V. Levchenko and V. Jackson). The deg-H2A is degraded H2A in which a 15 amino acid peptide of the C terminal has been proteolytically removed. When H2A, H2B is no longer present in a nucleosome, the C terminal region is sensitive to proteolysis [126] from a protease which is a minor contaminate in the NAPI preparation.

Laskey RA (2002) Efficient detection of biomolecules by autoradiography, fluorography or chemiluminiscence. Review 23, Amersham Biosciences... 

Thomson, J. and Torchia, D. A. 1984. Use of 31P nuclear magnetic resonance spectroscopy of 14C fluorography in studies of glycolysis and regulation of pyruvate kinase... 

Figure 3. Effect of UV light ( 3 10 nra) on incorporation of [3H]-2-nitroimipramine into human platelet membranes, as shown by SDS-PAGE and fluorography.

Figure 5. Dose response inhibition by imipramine of photolabeling of the 30 KDalton fraction in human platelet membranes in the presence or absence of varying concentrations of imipramine as indicated, followed by solubilization and SDS-PAGE/fluorography.

Fluorography of TLC Plates. TLC plates were developed in the appropriate solvent system and dried at 50° for 10-15 minutes. 

Fig. 1. Comparison ot human PR detected by photoaffinity labeling and immunoblotting. Intact T47D cells were incubated for 4 hours at (PC with 20 nM H]R5020. The cells were irradiated 2 min with 300 nm UV to photoaffinity label the receptors in situ, then homogenized, and a cytosol was prepared which was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were blotted onto nitrocellulose and PR in the right lane were visualized by immune analysis with MAb AB-52 radioactive proteins in the left lane were visualized by fluorography.

Figure 5-2 Informed consent for oral fluorography. (Reprinted with permission from the School of Optometry, University of Alabama at Birmingham.)...

KeUeyJS, Kincaid M. Retinal fluorography using oral fluorescein. Arch Ophthalmol 1979 97 2331-2332. 

Potter JW, Bartlett JD, Alexander LJ. Oral fluorography. J Am OptomAssoc 1985 56 784-792. 

In vitro pulse-chase experiments in the presence or absence of various inhibitors, the products are analyzed by immunoprecipitation, gel electrophoresis and fluorography. Transport is usually measured as the amount of precursor converted to mature form. In some cases, after labelling cells are fractionated and transport quantified by measuring the accumulation of immunoprecipita-ble polypeptides in the organelle. 

To estimate the amount of translation product in this case, an aliquot (5 il) is incubated in parallel in the presence of 5 [xM pSJmethio-nine in addition to the unlabelled methionine. The radioactive translation product is analyzed by SDS-PAGE and fluorography. The amount of S-labelled protein is calculated by incubating the isolated gel band with Opti-Fluor (Amersham) for 30 min and then measuring the radioactivity by scintillation counting. 

To measure binding, dialyzed translation mixture (diluted 5 times in HEPES medium) is added to Vero cells growing as monolayers in 12 well microtiter plates and kept at 24 C for 20 min in the presence of 1 mM unlabelled methionine and 10 jrM monensin (Moskaug ef al., 1988). To measure translocation, undiluted, dialyzed lysates are added to cells and kept for 20 min at 24 °C in the presence of 1 mM methionine (to reduce incorporation of any labeled methionine that had not been completely removed during dialysis) and 10 M monensin. The cells are then washed 3 times with ice cold HEPES medium and exposed to pH 4.5 for 3-5 min at 37 °C, treated with pronase and analyzed by SDS-PAGE and fluorography as described above. 

For in vitro labeling, 0.2 n-Ci pH]farnesyl pyrophosphate (Du Pont -New England Nuclear) and 2 ng of the CaaX-tagged protein is added to 20 il of reticulocyte lysate (Promega) as source of farnesyl transferase. The mixture is adjusted to contain 5 mM MgC. To carry out the full modification, 1 jxl of dog pancreatic microsomes (Promega), must also be added. The mixture is then incubated for 30 min at 37 °C. Labeled proteins can be recovered from the reaction mixture by immunoprecipitation with an appropriate antibody adsorbed to protein A-Sepharose, and analyzed by SDS-PAGE and fluorography (Wiedlocha etal., 1992). 

Analyse by immunoprecipitation, SDS-PAGE and fluorography. 19.8.7 Analysis of in vivo Farnesylation... 

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