A density gradient is formed in a centrifuge tube, and a mixture of proteins in solution is placed on top of the gradient. To identify the estradiol receptor, the protein mixture is first incubated with radioactive estradiol, which is readily detected. Only the estradiol receptor will bind to the steroid. Moreover, the steroid alone is too small to be influenced by the centrifugal force. After the centrifugation is complete, a small hole is made in the bottom of the centrifuge tube and portions of the gradient are collected and tested for radioactivity.

An antibody is a protein synthesized in response to the presence of a foreign substance called an antigen. The antibody recognizes a particular structural feature on the antigen called the antigenic determinant or epitope.

Any antibody-producing cell synthesizes antibodies that recognize only one epitope. Each antibody-producing cell thus synthesizes a monoclonal antibody. Any antigen may have multiple epitopes. The antibodies produced to the antigen by different cells are said to be polyclonal.

Immortal cell lines producing monoclonal antibodies can be generated by fusing normal antibody producing cells with cells from a type of cancer called multiple myeloma. A monoclonal cell line is isolated by screening for the antibody of interest.

A monoclonal antibody for the estrogen receptor can be isolated by searching for cell lines that produce an antibody that binds to the receptor. If an antibody for the receptor is present, it will bind to the receptor and alter the sedimentation constant of the receptor.

Once the monoclonal cell line is isolated, the antibody can be used to purify the estrogen receptor.

Antibodies are used as a reagent to determine the amount of a protein or other antigen present. Enzyme-linked immunosorbent Assay (ELISA) quantifies the amount of protein present because the antibody is linked to an enzyme whose reaction yields a readily identified colored product.

In western blotting or immunoblotting, proteins are separated in an SDS- PAGE gel, transferred to a sheet of polymer, and then stained with a fluorescent antibody.

A key step in understanding protein function is to determine the primary structure, or the amino acid sequence, of the protein. A preliminary step is to determine the amino acid composition of the protein. The protein is hydrolyzed, and the constituent amino acids are separated on an ion-exchange column. The amino acids are visualized by reaction with fluorescamine.

The amino acid sequence can be determined by Edman degradation. The protein is exposed to phenyl isothiocyanate (PTH), which reacts with the N-terminal amino acid to form a PTH-derivative. The PTH- amino acid can be released without hydrolyzing the remainder of the protein, and the degradation is subsequently repeated.

Because the reactions of the Edman degradation procedure are not 100% effective, it is not possible to sequence polypeptides longer than 50 amino acids. In order to sequence the entire protein, the protein is chemically or enzymatically cleaved to yield peptides of fewer than 50 amino acids. The peptides are then ordered by performing a different cleavage procedure in order to generate overlapping peptides.

Mass Spectrometry Can Be Used to Determine a Protein’s Mass, Identity, and Sequence Two mass spectrometry techniques can be used to determine a proteins mass: matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI). In MALDI, proteins are precipitated onto a matrix and a laser flash releases negatively charged ions. Time of flight (TOF) analysis is used to measure how rapidly the ions move toward a detector. Only picomoles or femtomoles of a protein are required for MALDI-TOF analysis.

Mass spectrometry allows determination of a protein’s identity. For instance, an unknown protein visible in a two-dimensional gel can be removed from the gel. The protein is then cleaved in some fashion and subjected MALDI-TOF, revealing a series of peptides with known masses. These peptide masses are then compared to proteins in a data base that are “electronically cleaved” by a computer using the same cleavage technique used to generate the protein fragments.

A proteins sequence can be determined with the use of tandem mass spectrometry.

1.Primary structures from different proteins can be compared to infer knowledge about structure and function. 2.Primary structure comparison of similar proteins from different species provides information about evolution. 3.Primary structure can be searched for internal repeats that may yield information on the history of the individual protein.

4.Primary structure can reveal the presence of amino acid sequences that regulate protein function and location. 5.Primary structure can provide insight into the molecular basis of disease. 6.Primary structure can be used as a guide to explore nucleic acid information.