Extraction of membrane proteins

Question 1

FFEM - freeze fracture electron microscopy

Answer 1

Consists of the study of membrane morphology and protein disposition.

The cell is preserved by a liquid nitrogen and the block is then fragmented.

The two membrane sheets separate and the membrane proteins remain attached to one or the other sheet

Question 2

WESTERN BLOT

Answer 2

It’s an eletroforese that uses fluorescently labeled antibodies to identify a specific protein.
This protein needs to be transferred from the polyacrylamide gel to a nylon membrane by electrotransfer (the protein has more affinity to the membrane than the gel)

Question 3

ELETROFORESE

Answer 3

• Uses SDS (ionic detergent) to extract the protein from the membrane
• The SDS treatment gives the denatured protein negative charges that are located around the primary structure.
• They put this protein in walls of a polyacrylamide gel (fibrotic network) and creates a electric field with the positive pole in the opposite part
• The molecule starts to migrates through the gel with a velocity that depends of their molecular size and weight.

Question 4

PURIFICATION

Detergents

Answer 4

The water soluble protein can be extracted from cytoplasm without lose their original conformation

já membrane proteins have hydrophobic parts that are attached with membrane lipids and this interactions need to be broken by a detergent, but when they are extracted they denature.

IONIC DETERGENT (SDS): denature
NON IONIC DETERGENT (TRITIAN-X-100): preserves protein structure