[[Membrane Potential Assignment[[
Membrane potential: The separation of charge across a membrane, measured in mV
Ungated channels (Na+, K+) are always open
Na+/K+ Exchange Pump is active, going against the gradient, keeping unequilibrium and net negative charge.
Neuronal Excitation: The firing of an action potential down the axon, caused by rapid redistribution of ions across the axonal plasma membrane.
Membrane proteins are amphipathic (part hydrophilic, part hydrophobic) to be able to work well with the tails, but also water/ions.
Facilitated diffusion needs no additional energy, ions pass through channels down their electrochemical gradient, which is determined by the charge of the ion as well as the concentration of the ion on each side of the membrane.
Equilibrium Potentials: The membrane potential at which diffusional and electrical forces on an ion are equal and opposite. When equilibrium potential is reached, there is no further net movement of the ions.
Calculating Eion, The Nernst Equation
What keeps Na+, K+, and Ca2+ from reaching equilibrium?
The Na+/K+ pump maintains an imbalance of Na+ and K+ across the membrane.
}}Typical concentrations inside and outside the neuron}}
Rate of ion movement, Ionic Driving Force
The Goldman Equation
]]Part 1: Acetylcholinesterase Assay (Week 1)]]
Objective: To determine which region of the brain (frontal cortex, striatum, hippocampus, and cerebellum) has the highest amounts of ACh.
Testing 4 regions of the brain: Frontal Cortex, Striatum, Hippocampus, and Cerebellum
The brain is homogenized with 0.1 M Phosphate Buffer (PB) to produce a 30 mg (homogenate)/ml (PB) solution.
Brain homogenate is the source of AChE (the enzyme) that breaks down ATChI into acetate and choline
Ellman Assay (above) quantifies the amount of ATChI broken down by the enzyme in the brain homogenate to determine which brain region of the 4 tested has the highest amounts of AChE/ACh.
Be able to calculate the volume of 0.1 M PB needed to produce a 30mg/ml homogenate.
After sonicating, place the homogenate on ice.
Materials
Performing the Ellman Assay
Calculations and Graphing
{{Part 2: AChE Inhibition Assay (Week 2){{
Performing the Assay
==Inhibited samples==
Calculations and Graphing:
Michaelis-Menten Plots
Lineweaver-Burk Plots
Convert your substrate concentration values and reaction rates to their reciprocal values to generate the Lineweaver-Burk plots.
Plot as a scatter graph with unconnected dots and add linear trendlines to each data set, showing the equation of each line on the graph. You will have to extend your axes and trendlines into the negative quadrants to be able to see where each line crosses the x-axis.
From the equations of the line, you can now calculate the Vmax and Km for the uninhibited and inhibited reactions.
Tacrine is a non-competitive inhibitor