in western blotting the non-specific sites on the membrane can be blocked by
Western blot Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100. Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands.[11] The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives.
Casparian strip The chemistry of the Casparian strip has been described as composed of suberin. According to some studies,[3] the Casparian strip begins as a localized deposition of phenolic and unsaturated fatty substances in the middle lamella between the radial walls, as partly oxidized films. The primary wall becomes encrusted with and later thickened by deposits of similar substances on the inside of that wall. The encrustation of the cell wall by the material constituting the Casparian strip presumably plugs the pores that would have otherwise allowed the movement of water and nutrients via capillary action along that path.[4] The cytoplasm of the endodermal cell is firmly attached to the Casparian strip so that it does not readily separate from the strip when the cells are subjected to contraction of the protoplasts. At the root, the casparian strip is embedded within the cell wall of endodermal cells in the non-growing region of the root behind the root tip.[5] Here, the casparian strip serves as a boundary layer separating the apoplast of the cortex from the apoplast of the vascular tissue thereby blocking diffusion of material between the two.[6] This separation forces water and solutes to pass through the plasma membrane via a symplastic route in order to cross the endodermis layer.[5]