Question

1) What properties would you consider important for designing a fluorescent probe that could be used in vivo to study an enzyme activity found in blood vessels?

2) What is the difference between an epitope tag and a fusion protein? Describe how you might image dynamics of centrioles in live cells during cell division assuming you use a genetic approach involving creating a fusion protein. Use diagrams and words to explain what proteins you would use to do this and how you would design your experiment.

3) Show two different chemical labeling methods you could use to label high quality antibodies you have obtained from a company by using small organic fluorophores purchased from a chemical supplier. Draw the chemistry occurring on the antibody.

Answer 1

(1) The applications of fluorescence techniques are geared towards clinical imaging. The majority of the work in this clinical area is based on single-photon near infrared (NIR) or two-photon measurements. The lower scattering propensity of the longer wavelengths of light allows the ability to image deeper into the tissue. There is a rich literature on FRET and quenching phenomena being applied to biochemical problems. Researchers have taken advantage of that for design of probes, however additional, significant ingenuity has gone into the overall probe design to improve other meritorious attributes including

• delivery
• signal integrity
• and enzyme selectivity

.The majority of applications have been to determine a particular protease activity. examples of applications in arthritis , ischemia , and cancer.

(2)

Epitope tagging is a technique in which a known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which an antibody is available, the technique makes it possible to detect proteins for which no antibody is available.protein tags are peptide sequences that are attached to proteins to facilitate easy detection and purification of expressed proteins. In addition, they can also be used to identify potential binding partners for your protein of interest.

Three commonly used design techniques include tandem fusion, domain insertion, and post-translational conjugation.

Tandem fusion

The proteins of interest are simply connected end-to-end via fusion of N or C termini between the proteins. This provides a flexible bridge structure allowing enough space between fusion partners to ensure proper folding. However, the N or C termini of the peptide are often crucial components in obtaining the desired folding pattern for the recombinant protein, making simple end-to-end conjoining of domains ineffective in this case. For this reason, a protein linker is often needed to maintain the functionality of the protein domains of interest

Domain insertion.

It involves the fusion of consecutive protein domains by encoding desired structures into a single polypeptide chain, but sometimes may require insertion of a domain within another domain.It is typically regarding as more difficult to carry out than tandem fusion, due to difficulty finding an appropriate ligation site in the gene of interest.

Post-translational Conjugation

In this technique protein domains fuses following ribosomal translation of the proteins of interest, in contrast to genetic fusion prior to translation used in other recombinant technologies.

(3)

NHS (Succinimidyl) Ester Method

In this method the conjugation of antibodies with widely available fluorescent dyes such as rhodamine derivatives occurs.NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. It is typically performed in a phosphate buffer with subsequent on-column separation from the unlabeled dye. The main disadvantage is that the esters are unstable because they are moisture-sensitive. The labeled antibody should be used immediately after the end of the reaction.

2. Isothiocyanate Method

You may used this method to make fluorescein isothiocyanate (FITC), which is very popular in the preparation of fluorescent proteins and antibodies. If you’ve worked with fluorescent microscopy, you’ve dealt with FITC. Isothiocyanate analogues of different standard dyes are also available.

Isothiocyanate is more stable than NHS but it is harder to make and your labeling reaction will likely be less efficient with this method. As with NHS, the excess dye should be removed after the reaction by chromatography.