1. 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

2. Membranes: selective barrier
REGULATE TRAFFIC OF IONS AND MACROMOLECULES

3. 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

4. Proteins in membranes contain CHO if outside of cell

5. Locations of proteins in the membrane

6. Locations of proteins in the membrane conference

7. BICONCAVE SHAPE of ERYTHROCYTES

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

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

11. SDS -
solubilizes
proteins and
gives them a
net negative
charge

12. SDS PAGE

13. 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

16. 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)

18. 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

23. 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