1. If purified p53 is to be concentrated in a Centricon-30 microconcentrator, recovery of the protein can be enhanced fivefold to sevenfold by using solutions that have been pre-treated with Chelex-100 from the time of lysis of the infected Sf9 cells and throughout the purification procedure.
2. Testing of other membranes both non-nylon (nitrocellulose, DEAE cellulose) and nylon (duralon, nytran) has shown that Nytran plus is far more effective in its retention of DNA 50-60 nucleotides in length.
3. If desired, it is possible to do a double blot in which the mobility shift gel is assembled with a piece of nitrocellulose BA-85 (Schleicher & Schuell, pore size 0.45 p) immediately on top of the gel, with a piece of Nytran plus membrane on top of the nitrocellulose membrane. In this case, pre-wet the nitrocellulose in 0.33X TBE for 15 min before assembling the blot. After blotting, wash the nitrocellulose with several changes of dH2O before immunodetection. It is also possible to store the membrane dry at this point if time is limited, and re-wet with dH2O later prior to proceeding with immu-nodetection.
4. For reproducible results, it is important to complete the full reaction of the proteins with IAA followed by IAM before freezing down the samples at -70°C, rather than discontinuous treatment where the samples are frozen down after treatment with IAA and then thawed to treat with IAM at a later point in time. The artefactual differences can be seen in the lower panel of Fig.9.2.
5. Migration of a specific protein in a urea-polyacrylamide gel cannot always be predicted on the basis of the molecular weight of the protein. For example, cytochrome c with a molecular weight similar to that of TRX actually migrates at a rate similar to that of p53, so it should be run on a 5.25% gel, rather than a 9% gel, to separate its redox forms.
6. For ECL detection of the Western, incubate the membrane in PBS + 0.05% NP-40 + 5% nonfat dry milk for 1 h at room temperature with gentle shaking. Then add the primary antibody diluted in PBS + 0.05% NP-40 + 0.5% powdered milk at the same concentration as specified for the Odyssey detection, and incubate for 1 h at room temperature with shaking. Wash the membrane four times with PBS + 0.05% NP-40 for 15 min/wash. Incubate the membrane with the secondary antibody, horseradish peroxidase-coupled goat anti-mouse IgG diluted 1:3,000 in PBS + 0.05% NP-40, for 1 h at room temperature: Remove unbound secondary antibody by washing the membrane four times with PBS + 0.05% NP-40 for 15 min/wash. To detect the bound HRPcoupled antibody, mix the two ECL reagents 1:1 just before use, place the membrane on a piece of Saran wrap protein side up and pipette on the mixed reagent so that the membrane is fully covered. Incubate for 1 min at room temperature, and then carefully lift the membrane with forceps to drain off the excess reagent and wrap the membrane in Saran wrap. Expose the wrapped membrane to a sheet of Amersham Hyperfilm-ECL in a film cassette for different periods of time, as judged by an initial exposure of 1 min.
7. For protein sodium dodecyl sulfate -polyacrylamide gel analysis, remove the 1X PBS from the centrifuged cells and resuspend the cell pellet in 200 pL of EMSA buffer A (20 mM HEPES, pH 7.6; 20% glycerol; 10 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 1 mM DTT; and 0.1%NP-40) with protease inhibitors (0.5 mM PMSF, 0.5 pg/mL leupeptin, 2 pg/mL aprotinin, and 0.7 pg/mL pepstatin A), until no clumps are present. Transfer to a large microfuge tube and leave on ice for 10 min. Then centrifuge at 5,000^ for 4 min at 4°C in a refrigerated microfuge, and transfer the cytoplasmic extract to a clean tube for storage at -70°C. Add 150 pL of EMSA buffer B (same as EMSA buffer A, but with 500 mM NaCl final concentration) with protease inhibitors (same as above) to each pellet, resuspend the pellet thoroughly, and leave on ice for 30 min. Centrifuge at 14,000 rpm for 15 min at 4°C, and carefully transfer the nuclear extract to a fresh tube and store at -70°C until analysis on sodium doceyl sulfate-polyacrylamide gels.
8. Scion Image for Windows (Scion Corporation) can be downloaded at no charge to the user. It is the Windows version of Scion Image and is similar to NIH Image, originally written at the National Institutes of Health for the Macintosh. NIH Image (1.62) is a public domain image processing and analysis program for the Macintosh that can also be downloaded for free.
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