Question

Which technique would you use to determine the following? You can only use each technique once. Not all techniques will be used. Explain your answer.

techniques available:

- SDS-PAGE and Western blot

- TLC

- freeze-fracture

- lactoperoxidase labeling

- FRAP/photobleaching

- photoactivation

- hydropathy plot

1. You want to determine if a membrane has proteins in it or not.

2. You want to determine if a cell membrane has the Na+/K+ transporter.

3. You want to determine the types of lipids in a given membrane.

4. You want to compare two membranes and see which is more fluid.

5. You want to determine if a protein is transmembrane or lipid anchored.

Answer 1

1. SDS PAGE and western blot: It is a technique used for identification of particular protein from the mixture of protein. In this technique labelled antibody is used for identifying the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.

2. lactoperoxidase labeling

3. TLC: A particularly frequent approach is to obtain information on the various phospholipid components of the lipid extract under investigation. This can be achieved easily and efficiently by subjecting a sample to thin-layer chromatography (TLC).

4. FRAP: Lateral motion of molecules within the membrane can be measured by a number of fluorescence techniques: fluorescence recovery after photobleaching (FRAP) involves photobleaching a uniformly labelled membrane with an intense laser beam and measuring how long it takes for fluorescent probes to diffuse back into the photobleached spot.

5. Hydropathy plot: It is used to characterize or identify possible structure or domains of a protein, whether it is transmembrane or lipd anchored.