Biomembrane and Cell signalling BCH 452 Dr. Samina Hyder Haq Assistant professor Dept of biochemistry Collage of Science King Saud university

Techniques used to study the biomembranes. By simple flourescent light microscopy. Study cells whole with electron microscopy. Cells are prepared through a process of fixing, embe dding, slicing, and staining.

How can we isolate membrane? 1. Vigorous Homogenisation of tissues. 2. Differential centrifugation using sucrose density gradient

Experimental Arrangement for the Study of Planar Bilayer Membrane. A bilayer membrane is formed across a 1- mm hole in a septum that separates two aqueous compartments. This arrangement permits measurements of the permeability and electrical conductance of lipid bilayers.

Permeability Coefficients (P) of Ions and Molecules in a Lipid Bilayer.

Freeze etching microscope freeze-etch electron microscopy, used to examine either the exterior or interior of cells. In this technique, the frozen block is cracked with a knife blade. ice level is lowered around the cells (and to a lesser extent within the cells) by the sublimation of ice in a vacuum as the temperature is raised—a process called freeze-drying. The parts of the cell exposed by this etching process are then shadowed as before to make a platinum replica. This technique exposes structures in the interior of the cell and can reveal their three-dimensional organization with exceptional clarity

Freeze fracture technique provides a way of visualizing the interior of cell membranes. Cells are frozen and then the frozen block is cracked with a knife blade. The fracture plane often passes through the hydrophobic middle of lipid bilayers, thereby exposing the interior of cell membranes. The resulting fracture faces are shadowed with platinum, the organic material is dissolved away, and the replicas are floated off and viewed in the electron microscope. Such replicas are studded with small bumps, called intramembrane particles, which represent large transmembrane proteins. The technique provides a convenient and dramatic way to visualize the distribution of such proteins in the plane of a membrane.

Freeze fracture technique

The Preparation Of red blood Cell Ghost

SDS –PAGE of Erythrocyte membrane When the plasma membrane proteins of the human red blood cell are studied by SDS polyacrylamide-gel electrophoresis, approximately 15 major protein bands are detected, varying in molecular weight from 15,000 to 250,000 daltons. Three of these proteins spectrin, glycophorin, and band 3 account for more than 60% (by weight) of the total membrane protein

Analyses of protein on SDS-PAGE

The use of mild detergents for solubilizing, purifying, and reconstituting functional membrane protein systems

SDS –PAGE of erythrocytes

The major ones are referred to as bands 1,2,3,4.1,4.2.5,6 and 7 Staining with the periodic acid-sciff(PAS) reagent brings out several protein that are rich in carbohydrates.These bands are desighned as PAS- 1,PAS-2,PAS-3, PAS-4 The protein corresponding to bands 1,2,4.1,4.2,5 and 6 can be extracted from the membranes by altering the ionic strength or pH of the medium and so they are peripheral proteins. When Sealed ghost or intact RBC are treated with proteolytic enzymes there is no effect on them, which shows that they are present inside the cytoplasmic side of the cells. When leaky ghost of RBC are treated with proteiolitic enzymes they are extensively digested.

Band 6 is glyceraldeyde 3-phosphate dehydrogenase,a glycolytic enzyme. Wheras band 5 is actin. A key protein in muscle contraction and cell motility. Bands 1 and 2 called spectrin, associciate to form an extended filamentous network. Spectrin in concert with other proteins probably stabilizes the regulatory shape of the red blood-cell membrane. In contrast bands 3 and 7 and all four PAS bands can be dissociated from the red-cell membrane only by detergent or organic solvents. Hence they are integral membrane protein.This is further proved by freeze fracture technique

Erythrocyte Cell membrane

If you expose an intact cell to a protease enzyme, you can shave off the protein found on the outside of the cell. If the cell is broken, you can remove proteins from the inside of the membrane as well, leaving only the parts of proteins within the cell wall intact.

Different functions of integral membrane proteins