By Samols D., Agrawal A., Kushner I.
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17. 18. 19. 20. 21. 22. TEMED. Glycerol. 25% Isopropanol, 7% glacial acetic acid (fixer/destainer). 1% Coomassie brilliant blue in fixer/destainer. 8, 6 mL H20, bromophenol blue to color. Dithiothreitol (DTT). 2% Agarose. , Piscataway, NJ, or equivalent). PVDF (nylon) membranes. Methanol. 2M NaH2P04 stock in 900 mL HzO. Filter paper for blotting (Whatman #l, or equivalent). Distilled water (dHaO). 3. 1. 2. 3. 5 pL 150 & 3. 1. Sample Solubilization Boil samples in sample solubilization buffer for lo-30 min.
Commun. 63,378-3&I. 7. Hardison, R. and Chalkley, R. (1978) Polyacrylamide gel electrophoretic fractionation of histones, in Methods in Cell Biology, vol. , and Kleinsmith, L. ), Academic, New York. 8. Hrkal, Z. (1979) Gel-type techniques, in Electrophoresis. ), Elsevler, Amsterdam 9. Alfageme, C. , and Cohen, L. H. (1974) Histones of Drosophila embryos. Electrophoretic isolation and structural studies J. Biol. Chem. 249,3729-3736. 10. Gabriel, 0. and Gersten, D. M. (1992) Staining for enzymatic activity after gel electrophoresis.
Degas this mixture under vacuum for 30 s and then add TEMED (12 pL). Pour off the water overlayering the gel and wash the gel surface with about 2 mL of stacking gel solution and then discard this solution. The gel slab is now filled to the top of the plates with stacking gel solution and the well-forming comb placed in posrtron (see Chapter 5). Walker 38 12. When the stacking gel has set (-15 min), carefully remove the comb. The gel is now ready for running. The conditions of running and sample preparation are exactly as described for SDS gel electrophoresis m Chapter 5.
Acute Phase Proteins by Samols D., Agrawal A., Kushner I.