#streptavidin

Streptavidin conjugate is stable and water-soluble fluorescent proteins derived from cyanobacteria and Eukaryotic algae. In these organisms they are used as accessory or antenna pigments for photosynthetic light collection. They absorb energy in portions of the visible spectrum that are poorly utilized by chlorophyll and through fluorescence energy transfer, convey the energy to chlorophyll at the photosynthetic reaction centre.

This interview was based on an in-class interview with Jessica, who is a doctoral student in molecular toxicology and is studying the effects of an herbicide, Acetochlor, on mice; and also uses “click chemistry” to study the structure and constituents of biomolecules.

As an herbicide, Acetochlor is used primarily on corn, but also soy and peanuts, and less often on other crops. The world divides into Acetachlor “targets,” which are unwanted plants--weeds--that compete with marketable crops farmers are growing.  Any non-target organism, plant or animal, is referred to as an “off-target” organism. 

Little is known about the long-term effects of Acetochlor on off-target organisms, such as human beings. We do know that Acetachlor preferentially binds to cysteine, one of the twenty amino acids that are the building blocks of proteins.  A common experiment is to inject mice with Acetochlor and after four hours, add a molecular probe, which binds with reactive cysteine so that the effects of the Acetochlor may be studied.

Not all cysteines are reactive, but many are.  The effect of large doses of Acetochlor is to render metabolic pathways nonfunctional if enough Acetochlor binds to reactive cysteine.  Pathways may also become nonfunctional if the dose of Acetochlor is lower but there are more reactive cysteines.

Acetochlor has affinity for enzymes that are involved in fatty acid oxidation, the process by which animals metabolize fatty acids for energy when glucose is unavailable.  Thus, the adverse effects of Acetochlor become apparent when animals rely on fatty acids for essential metabolic energy. 

Click chemistry is a method for analyzing the composition of biomolecules and depends on the use of probes.  Probe molecules have two active regions at opposing ends:  1) the warhead, which is the binding site of the probe; and 2) the alkyne, which is a nitrogen triple bond, N≡N. 

If we bind rhodamine with a probe to a biomolecule of interest, we can learn about its composition by pulling it through a gel to which we apply an electric current.  The current separates the components of the molecule on the basis of its weight as done in gel electrophoresis.

R-PE belongs to the family of phycobiliproteins, which are derived from cyanobacteria and eukaryotic algae. R-PE has absorption maxima at 496, 546, and 565 nm respectively and emission at 578 nm, allowing it to be detected in the red channel by flow cytometry using 488 nm excitation. Antibodies are supplied at 2 mg/mL in PBS pH 7.4 containing 50% glycerol, 2 mg/mL BSA (IgG- and protease-free) and…