Question

Describe how FRAP is used to measure the movement of lipids and proteins within the bilayer. When examined by FRAP, certain integral membrane proteins are significantly less mobile than others. What accounts for this reduced mobility?

Answer 1

The FRAP or fluorescnce recovery after photobleaching is a high throughput screening technique which is based upon recording and quantifying the recovery of fluorescence in a biological sample after it has been intentioanlly bleached after a strong beam of light. This causes a bleaching effect in the sample and the excess of fluorescence dye re-enters the sample. This causes re-establishment of a fluorescnece emittance from the sample, or flurescence recovery. This fluoresence is recorded in a recording system and hence, the extent of this intensity represents the mobility of the sample.

This technique can be used to determine the mobility of lipids and proteins within the bilayer in following manner:

1. The whole plasma membrane is labelled with a fluorescence dye.
2. This fluorescence dye is recorded as base-line level.
3. The plasma membrane is exposed to a strong beam of light in small flashes to cause photobleaching at some pre-defined locations.
4. This causes a loss in fluorescence and the signal intesity drops down.
5. Since the plasma membrane if excessively loaded with flourescence dye, it again gets stained with the dye and fluorescence signal re-establishes or recovers.
6. This causes an increase in the fluorescence signal.
7. This change i.e. loss and gain in fluorescence signal represents the mobility of the plasma proteins and lipids.

Hence, in this way, the mobility and kinetics of the plasma proteins and lipids can be investigated. This also suggests that some proteins and lipids are more mobile than others since some of the locations of the plasma membrane show less fluorescence signals as compared to other locations. The fluidity and large amount of structural account for this mobility.