Studying Membrane Transport Flashcards
Method to study Bulk Transport where long exposure of light to a fluorescently tagged cell area causes it to lose color/fluorescence. If that protein is mobile, then the color should return. Basically a technique to see if proteins are found in vesicles and are mobile.
Fluorescence Recovery After Photobleaching
See PDF for figures:
Acquired images for the recovery of fluorescently labelled vesicles after application of ML-9, before bleaching (8 sec), immediately after photobleaching (10 sec) and at 16, 48 and 120 sec. Bleached areas are indicated by white arrows.
Quantification of FRAP at several doses of ML-9 was done.
Results showed less recovered fractions of initial fluorescence. What does this mean?
ML-9 inhibits vesicle trafficking
Method where in membrane disruption is done by adding a mild detergent to solubilize membrane proteins and separate out lipids and membrane proteins, purification, isolation, and membrane reconstitution with only the transport protein of interest. This can then be used to study different transport rates.
Membrane Reconstruction (Succeeding tests done through dye or pH-based assays)
Fura-2, AM is a high affinity, intracellular calcium indicator. Higher fluorescence means more calcium in the cell. What happens to Fura-2, AM fluorescence when blocking specific transport proteins? For example the Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA)?
If a transport protein is blocked, that means that calcium will not be able to move out meaning higher concentration of Ca and higher fluorescence. If lower fluorescence, this could imply that it is promoting transport proteins
Differences in the concentrations of ions on opposite sides of a cellular membrane dictating the voltage of the cell
Membrane Potential, approx +40 mV to -70 mV
How is repolarization achieved during action potential?
From depolarization due to opening of Na+ channels, voltage insensitivity is reached. Once this is reached Na+ channels close and K+ channels open to return it to -70mV
Why is Action potential important?
This is to make sure that signals are transferred only one way allowing of movement of impulses across nerve cells.
What happens when action potential reaches the nerve terminal
Opening of voltage-gated Ca+2 channels at the presynaptic terminal causing release of neurotransmitters
How to read a patch clamp?
Oscilloscope trace shows the current passing in circuit, it will usually move up and down and lets you read the voltage.
Two types of patch clamp
Current Clamp and Voltage Clamp
Type of clamp where change in membrane potential (voltage) is measured.
Current clamp (current is constant)
Type of clamp where change in membrane current is measured.
Voltage clamp (Membrane potential/voltage is kept constant)
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