By Jamila I. Horabin
Meant for Molecular Biologists, Biochemists, and Geneticists drawn to manipulating and studying the Hedgehog Signaling Pathway, this quantity deals numerous specific chapters on experimental description, together with anecdotal tips and notes.
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Extra resources for Hedgehog Signaling Protocols (Methods in Molecular Biology Vol 397)
Are indications that Shh diffuses across the chick limb, as indicated by a cell differentiation assay (18), while Shh protein has been detected by immunohistochemistry (19). Nevertheless, cells expressing membrane tethered Shh still induce complete digit duplications (17). Here, we will describe how to apply Shh protein on beads and graft pellets of Shh-expressing cells to chick limb buds. Each method has its own merits; cells process the Shh protein, while Shh-soaked beads can be readily removed and thus permit experiments that investigate the importance of the timing of Shh signaling (17).
And Lee, J. (1982) Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nature 296, 564–566. 12. , and Alberts, B. (1984) Microcontrolled release of biologically active compounds in chick embryos: beads of 200-microns diameter for the local release of retinoids. Anal. Biochem. 142, 542–555. 13. , and Eichele, G. (1985) A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development. Dev. Biol. 109, 82–95. 14. , and Alberts, B.
13. Do not vortex, shake, or agitate vigorously and avoid the formation of bubbles. We recommend mixing by gentle inversion. 14. 0. 15. cm/h = flow rate (mL/h) divided by the cross-sectional area of the column (cm2). 16. During the dialysis of myristoylated ShhN, a precipitate may be visible although the majority of the protein remains in solution. 17. 5 mM DTT to remove the octyl-G-Dglucopyranoside since the proteins remain reasonably soluble in the absence of the detergent. 5 mM DTT containing 1% (w/v) octyl-G-D-glucopyranoside since it is insoluble in the absence of the detergent.