tutorial 4: Western Blot Day 3 antibody-based protein detection

how does SDS bind to proteins

• SDS strongly binds to the positively charged and the hydrophobic residues of proteins through its sulfate groups and alkyl chains, respectively

when SDS meets a protein what happens?

SDS’s hydrocarbon tail dissolves any hydrophobic
region of the protein, while the sulfate end breaks non-covalent ionic bonds. This causes the protein to lose its secondary and tertiary structure, and unfold

purpose of this lab/experiment

This lab will involve the extraction of cellular proteins form 2 different E. coli strains and identification of the Beta-galactosidase producing strain by means of SDS-PAGE and an anti-beta- galactosidase monoclonal antibody.

why are Polyvinylidene (PVDF) difluoride membranes widely used for immunoblotting techniques such as Western blots

PVDF membranes has high protein binding capacity

monoclonal antibodies

• (mAbs)

are generated by identical B cells which are clones from a single
parent cell. This means that the monoclonal antibodies have monovalent affinity and only recognize the same epitope (antibody binding site) of an antigen (a substance that stimulates immune response).

• Monoclonal antibodies are produced ex vivo using tissue-culture techniques

polyclonal antibodies

• (pAbs) are mixture of heterogeneous which are usually produced by
  different B cell clones in the body. They can recognize and bind to many different epitopes of a single antigen.

• Polyclonal antibodies are produced by injecting an immunogen into an animal

what is the secondary antibody in this experiment?

• anti-mouse IgG alkaline-phosphatase (AP) conjugate.

• This complex is an antibody which recognizes the mouse IgG antibody
  and has the alkaline phosphatase enzyme covalently linked to it

what will the protein-antibody-antibody-alkaline-phosphatase-complex react with?

BCIP (5-bromo-4-chloro-3-indolyl-phosphate) which is a substrate of alkaline
phosphatase in conjunction with NBT (nitroblue tetrazolium) for the color detection of alkaline phosphatase activity (a purple-grey color on the filter).

• BCIP (5-bromo-4-chloro-3-indolyl-phosphate) is a substrate of alkaline phosphatase in conjunction with NBT (nitroblue tetrazolium)

what is TBST? what does it do?

tris (tris(hydroxymethyl)aminomethane)-buffered saline and Tween 20 (non-ionic
surfactant used as a detergent); a buffer for washing nitrocellulose membranes in Western Blotting.

• Tween 20 is used as a washing agent in immunoassays. It prevents non-specific binding of other proteins (e.g. antibodies used in the detection system) to the surface of the membrane.