Conference on Membranes The objective of the conference is to answer a series of questions using several techniques designed to measure characteristics of membrane proteins. The objective of this video is to describe several techniques designed to measure characteristics of membrane proteins. Membranes are semipermeable

Membranes: selective barrier REGULATE TRAFFIC OF IONS AND MACROMOLECULES

Assumptions for the membrane conference Proteinases, labeling system, and other protein- or salt-containing treatments usually only act on the proteins exposed to the outside of vesicles as these systems do not normally cross the hydrophobic region of vesicle membranes Solution (protein solubilized in solvent) or remaining proteins maybe subjected to SDS PAGE Alterations of exposed proteins during treatment are detected as missing bands/or selectively labeled bands in SDS PAGE

Proteins in membranes contain CHO if outside of cell

Locations of proteins in the membrane

Locations of proteins in the membrane conference

BICONCAVE SHAPE of ERYTHROCYTES

Leaky Right-side-out or intact In-side-out

EXTRACTION PROCEDURES HIGH SALT CONCENTRATION or pH REMOVES PERIPHERAL PROTEINS DETERGENTS TRITON X 100 - NON-IONIC BREAKS BONDS BETWEEN LIPID AND PROTEINS MAKES VESICLES LEAKY SDS - IONIC DETERGENT EXTRACTS ALL MEMBRANE PROTEIN SOLUBILIZES PROTEINS for SDS PAGE

SDS - solubilizes proteins and gives them a net negative charge

SDS PAGE

SDS PAGE Molecular weight is detected by comparing the migration Proteins of known size in a standard are run in the same gel as are the unknowns Molecular weight is detected by comparing the migration of the unknown protein with the migration of proteins of known weight in the standard

LABELING TECHNIQUES LACTOPEROXIDASE and I125 LACTOPEROXIDASE - ATTACHES TO PROTEINS OUTSIDE OF MEMBRANE THAT HAVE TYROSINE RESIDUES ATTACH I125 WILL MAKE IT RADIOACTIVE Visualized with fluorography (autoradiography)

LABELING TECHNIQUES LECTINS - BINDS TO CARBOHYDRATES MICROSCOPY GEL ELECTROPHORESIS AFFINITY CHROMATOGRAPHY PROTEOLYTIC ENZYMES - REDUCE SIZES OF PROTEINS ALTERS NUMBER OF BANDS AND LOCATION ON THE SDS-GEL IMMUNOCYTOCHEMISTRY -- ANTIBODIES BIND AND LABEL SPECIFIC PROTEINS

Approach 1. Set up the experiment with different types of vesicles leaky intact Right-side-out or intact Approach In-side-out 1. Set up the experiment with different types of vesicles leaky -- also produced by triton X 100 treatment right-side out (intact) inside-out vesicles 2. Treat vesicles, run gel electrophoresis (SDS PAGE) to measure size of protein based on molecular weights 3. Use fluorography to detect presence of radioactivity 4. Use carbohydrate-labeling system if appropriate to detect sugars 5. Use immunocytochemistry to detect specific proteins