Recombinant autoradiography

For MK2, one may use a peptide substrate (see, e.g., Manke et al, 2005) or recombinant heat shock protein (hsp) 25 (Stokoe et al, 1992). For the peptide substrate, analysis is as described for the p90RSK assay. If using hsp27 as substrate, samples of the reaction products are analyzed by SDS-PAGE and autoradiography or quantitation performed by phosphorimager. 

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. 

Providing all the reagents, solutions and apparatus are at hand a set of 2-4 M13 recombinant DNA samples can be sequenced in an 8 hour working day. When using fresh a-32P dATP label overnight autoradiography is often sufficient and if all is well sequences totalling 500-1000 nucleotides may be determined daily. 

The concentration of each C3 protein preparation is determined with a Biorad protein assay kit, using BSA as a standard. The relative activities of C3 transferase preparations can be determined by ADP-ribosylation of recombinant Rho protein or by microinjection. For the ribosylation assay, 10 ng of recombinant RhoA protein are incubated for 1 h at 37 °C in 50 1 of ADP-ribosylation buffer (50 mM Tris-HCI, pH 7.4, 50 mM NaCI, 5 mM MgCl2, 0.3 mM GTP, 1 mM DTT, 1 iCi [ PJNAD) with varying amounts of C3 protein, ranging from 1 to 200 ng. Gel electrophoresis sample buffer is added, the samples are boiled, and proteins resolved by SDS-polyacrylamide gel electrophoresis (13.5 %). Protein is first visualized with Coomassie blue, and then the gel is destained with many washes to remove any unincorporated [ PjNAD. ADP-ribosylated rho is visualized by autoradiography. 

Fig. 1. Time course of ADP-ribosylation of Rho. 1 ng of recombinant GST-Rho (fusion protein) was incubated with 20 (iM of [ P]-NAD and 3ng of the wild type ( ) or the E173Q (o) mutant of C3 exoenzyme in a total volume of 50 nl of ADP-ribosylation buffer, as described in the text, at 30°C for times indicated. The reaction was terminated by the addition of trichloroacetic acid and Na deoxycholate. After centrifugation, the pellets were subjected to SDS polyacrylamide gel electrophoresis, followed by staining with Coomassie Brilliant Blue and autoradiography. P-ADP-ribosylation of Rho was quantified by cutting out the radiolabeled GST-Rho bands and determining their radioactivities. The relative ADP-ribosylation was expressed as the ratio of the P incorporation into GST-Rho at each point to the maximal P incorporation by the wild type C3 exoenzyme... 

In another approach, the colony hybridization technique (Figure 18D, bacteria are screened by using a radioactively labeled nucleic acid probe, an RNA molecule or a single-stranded DNA molecule with a sequence complementary to that of a specific sequence within the recombinant DNA. Bacterial cells are plated out onto solid media in petri dishes and allowed to grow into colonies. Each plate is then blotted with a nitrocellulose filter. (Most of the original colonies remain on the petri dishes.) The cells on the nitrocellulose filter are lysed, and the released DNA is treated so that hybridization with the probe can occur. Once nonhybridized probe molecules have been washed away, autoradiography (Biochemical Methods 2.1) is used to identify the colonies on the master plate that possess the recombinant DNA. 

For biological studies (e.g., autoradiography, morphology, immunofluorescence staining, cell transformation, recombination) cells are grown on either a culture dishes or on glass slides (5x1 cm) which have a grid imprinted for better identification of the cells after microinjection (see Fig. 3). 

Clones containing genomic DNA of a particular sequence may be identified by plaque hybridization, which is similar in principle to colony hybridization for detection of particular plasmid recombinants. A lawn of E. coli containing several thousand plaques of independent recombinants is blotted on to a nitrocellulose filter. The DNA is released from the blotted recombinant phage, denatured by alkali, and neutralized. A radioactive probe of nucleic acid containing sequences of the gene of interest is then hybridized to the DNA plaques on the filter. Positive plaques, detected by autoradiography as radioactive DNA-RNA or DNA—DNA hybrids, are then picked off and amplified at lower plaque density (approx. 100 per plate). 

The DAG kinase assay is currently the most commonly employed biochemical method for the quantification of total cellular DAG. This assay utilizes recombinant bacterial DAG kinase to phosphorylate DAG that has been extracted from the cell with the method of Bligh and Dyer (1959). Utilization of p P] ATP as a source of phosphate allows the direct detection of P-labeled DAG via autoradiography and scintillation counting. The DAG kinase assay is linear over a range of 25 pmol to 20-25 nmol of DAG, and is very useful for the elucidation of biochemical pathways (Kennerly et al, 1979 Preiss et al, 1987). The only drawback to this approach is its inability to identify individual DAG species. 

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