By David M. Terrian
A suite of comfortably reproducible experimental tools for learning the subversion of standard mobile signaling that results in melanoma. provided in step by step aspect to make sure winning effects, the tools contain confirmed options for the research of apoptosis and mobile loss of life, complementary protocols for manipulating and/or tracking oncogenic indications in melanoma cells, and strategies for learning protein-protein interactions. a close protocol for taking pictures natural samples of malignant cells from frozen tissue specimens and substitute suggestions for examining their genomic DNA can also be defined.
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Additional resources for Cancer Cell Signaling. Methods and Protocols
Following centrifugation at 15,000g for 5 min in a Eppendorf-style centrifuge, the supernatants were collected, quantitated using a Bradford microtiter assay, and prepared in a reducing 28 Hayward, Wang, and Day Fig. 4. Verification of Rb−/−PrE genotype. Primer sets 025-026 (Rb+/+) and 025-027 (Rb−/−) amplified only wild-type (wt) alleles (400 bp) from wild-type control cells (Rb+/+). The same primer sets amplified wild-type (400 bp) and mutant alleles (420 bp) from heterozygous control cells (Rb-/+).
The dissection of the embryonic urogenital sinus is too lengthy to be discussed in detail here. The specific protocol and diagrammatic depiction of the dissection including the location of cuts and the appearance of the various products are illustrated in ref. 34. 2. Following dissection, the urogenital sinuses should be submerged in a 10 mg/mL solution of 1:250 trypsin in calcium/magnesium-free Hanks solution for approx 75 min either on ice or at 4ºC. Tryptic digestion is then terminated by washing the sinuses three times in medium containing 10% FBS.
Oligo-dT cellulose, Type 3. 10. Glyoxyl, 40% solution and dimethyl sulfoxide (DMSO). 11. 8. 12. Biotrans nylon membrane (ICN, Costa Mesa, CA). 13. , Arlington Heights, IL). 14. , Madison, WI). 15. 0. 16. Denhardt solution 100X: 2% Ficoll 400, 2% polyvinylpyrrolidone, 2% bovine serum albumin (BSA) in double-distilled H2O (dd)H2O. 17. Hybridization solution: 5X standard sodium citrate (SSC), 5X Denhardt solution, 1% sodium dodecyl sulfate (SDS), and 100 µg/mL salmon sperm DNA. 18. 1% SDS. 19. 0. 20.