Western Blotting

Fiber pads

________: press the gel and nitrocellulose together tightly and uniformly, eliminate air bubbles, and allow efficient transfer of proteins out of the gel and onto the membrane.

Blocker

________: prevents the primary antibody from randomly adhering to the membrane and obscure or weaken the specific antibody signal.

secondary antibody

The enzyme that is linked to the ________ oxidizes the colorimetric substrate into an insoluble colored precipitate, leaving a visible deposit on the membrane at the precise location of the blotted myosin light chain proteins.

foreign invaders

Antibodies: key proteins found in all animal immune systems that detect ________ such as viruses, bacteria, and allergens and tag them for destruction.

electrophoresis

Proteins separated by ________ are then transferred or "blotted "from the gel onto a paper- like membrane.

buffer

Blotting ________: contains tris to maintain pH, glycine ions to transmit current, and ethanol to facilitate protein binding to the nitrocellulose.

Western blotting

________ can categorically identify a specific protein among hundreds or thousands of other proteins within biological samples.

SDS

Proteins, still negatively charged from the ________, migrate out of the gel and bind to the surface of the membrane, creating a mirror image of proteins separated in the original gel.

sample of cells

First, proteins are extracted from a(n) ________ or tissue.

Mini Tetra Blot apparatus

________: the Mini Tetra- Blot is specifically designed to pass electric current horizontally through the gel, forcing the negatively charged proteins to migrate out of the gel onto the nitrocellulose membrane.

fiber pads

Blotting paper (or Filter paper): used to support the gel and nitrocellulose and to protect them from the ________ during assembly and electrophoresis.

Western blotting

________: an immunodetection technique used by proteomic scientists to detect and quantify specific proteins in complex biological samples.

Western blotting

an immunodetection technique used by proteomic scientists to detect and quantify specific proteins in complex biological samples

Antibodies

key proteins found in all animal immune systems that detect foreign invaders such as viruses, bacteria, and allergens and tag them for destruction

Western blotting is based on two distinguishing features of proteins

molecular mass and antibody binding specificity

Mini Tetra-Blot apparatus

the Mini Tetra-Blot is specifically designed to pass electric current horizontally through the gel, forcing the negatively charged proteins to migrate out of the gel onto the nitrocellulose membrane

Nitrocellulose membranes

nitrocellulose acts as a solid support for proteins bound to its positively charged surface

Blotting paper (or Filter paper)

used to support the gel and nitrocellulose and to protect them from the fiber pads during assembly and electrophoresis

Fiber pads

press the gel and nitrocellulose together tightly and uniformly, eliminate air bubbles, and allow efficient transfer of proteins out of the gel and onto the membrane

Blotting buffer

contains tris to maintain pH, glycine ions to transmit current, and ethanol to facilitate protein binding to the nitrocellulose

Blocker

prevents the primary antibody from randomly adhering to the membrane and obscure or weaken the specific antibody signal

the primary antibody

binds only to the proteins

the secondary antibody

enzyme linked and bind specifically to the primary antibody.

The enzyme that is linked to the secondary antibody oxidizes the colorimetric substrate into

an insoluble colored precipitate, leaving a visible deposit on the membrane at the precise location of the blotted myosin light chain proteins.

nitrocellulose membranes

nitrocellulose acts as a solid support for proteins bound to its positively charged surface.