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Analysis Biomolecular Interactions of EcFrmR Protein with frmRAB Promoter DNA (CAT#: STEM-MB-0214-CJ)
Introduction
The frmRAB operon codes for: a regulator, FrmR (EcFrmR); a formaldehyde dehydrogenase, FrmA; and an S-formylglutathione hydrolase, FrmB. In E. coli, and many other organisms, formaldehyde in the cytosol reacts with the major reductant glutathione, yielding S-hydroxymethylglutathione, which is then oxidized by FrmA to S-formylglutathione. In E. coli S-formylglutathione is hydrolyzed to formate and glutathione by the product of the final gene of the frmRAB operon, FrmB, or by a second, less-efficient hydrolase YeiG11; some bacteria, such as the closely related Salmonella enterica serovar Typhimurium, lack frmB but possess yeiG. The proteins coded by the frmRAB operon and yeiG can permit bacteria to sense and detoxify formaldehyde.
Principle
Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.
Applications
Biochemistry
Procedure
1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.
Materials
• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Vector pET-22b, Escherichia coli BL21 cells
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Vector pET-22b, Escherichia coli BL21 cells
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