By James D. Stockand (Editor), Mark S. Shapiro (Editor)
The varied purposes during this quantity variety from the research of allosteric legislation of ion channel task utilizing a vintage mutagenesis technique, to the research of channel subunit stoichiometry utilizing a singular biophysical method according to fluorescence resonance strength move. Highlights contain tools for heterologous expression of ion channels in cells, for settling on channel structure-function, and for learning channel law.
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Additional info for Ion Channels: Methods and Protocols (Methods in Molecular Biology Vol 337)
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Spin 4 min in a clinical centrifuge to pellet cells. Carefully pour off the supernatant from the previous spin. Add 5 mL DMEM and triturate until cells are fully resuspended (taking care to avoid air bubbles). Add another 5 mL DMEM for a total volume of 10 mL. Spin 4 min in the clinical centrifuge to pellet cells. Carefully pour off the supernatant. Resuspend all cells in 10 mL RNase-free PBS (combining cells from multiple flasks). Place a drop of the cell suspension on a hemocytometer and count cells.
1. Outline of the basic procedure of the transfection of cultured neurons using the biolistic particle delivery system (PDS)-1000/He. 32 Gamper and Shapiro microcarriers to settle for 1 min. Pellet coated particles by briskly centrifuging and remove and discard the supernatant. Add 150 µL 70% ethanol to the pellet without resuspending it and discard wash (ethanol supernatant) without disturbing the pellet. Repeat wash step with 150 µL 100% ethanol and gently resuspend coated microcarriers in 10–15 µL 100% ethanol.
3. Production of virions: incubate transfected BHK cells for 48 h at 31°C and harvest the medium (which contains the released virions). Growing the cells at 31°C after electroporation increases the titer of the virion stocks. Freeze virion stocks overnight at −20°C, then store at −80°C. 4. 5 mL virion stock, add 50 µL chymotrypsin and incubate for 40 min at room temperature. After 40 min, add 55 µL aprotinin and incubate for 5 min at room temperature. Store activated virion stock at −20°C for up to a month (see Note 7).