Why List Applications? — These applications include a few papers, posters, protocols and helpful tips for your convenience in identifying an application which may be important to you. As a starting point, we are listing a few applications and reference papers which will give you complete protocols.
Antibody-Antigen Interactions:
Jolley ME, et al. (1995). The development of a fluorescence
polarization assay for the detection of antibodies to the O-polysaccharide
of Brucella abortus in bovine sera. AAVLD 38th Annual Meeting Abstracts,
120.
DNA/DNA Interactions:
DNA/RNA Interactions:
Wang C-HJ, et al. (1992). Detection of DNA/RNA by fluorescence
polarization. World Patent No. 9218650.
Garman AJ, Moore RS (1990). Detecting nucleic acid sequence using fluorescence
polarization by contacting homogeneous soln. With fluorescent nucleotide
probe capable of hybridising to nucleic acid sequence. European Patent No.
382433.
DNA Binding Proteins:
Walker GT, Linn CP, Nadeau JG (1996). DNA detection by strand
displacement amplification and fluorescence polarization with signal enhancement
using a DNA binding protein. Nucleic Acid Research 242, 348-353.
5', 3', or Internal Labeling of DNA is preferred
DTAF is superior to FITC
Make the linkage as short and rigid as possible
RNA Binding Proteins:
Drug Level Monitoring:
Jolley ME (1981). Fluorescence polarization immunoassay for
the determination of therapeutic drug levels in human plasma. J Anal Toxicol
5, 236.
Drug/Receptor Interactions:
Domain/Receptor Interactions:
Schindler U, Wu P, Rothe M, Brasseur M, McKnight SL (1995).
Components of a STAT Recognition Code: Evidence for two layers of molecular
selectivity. Immunity 2, 689-697.
Cell Membranes:
Protein Aggregation:
Micro Viscosity:
Hydrolytic Enzymes:
Ligand/Receptor Binding:
Kauvar LM, Higgins DL, Villar HO, Sportsman JR, Engqvist-Goldstein
A, Bukar R, Bauer KE, Dilley H, Rocke DM (1995). Predicting ligand binding
to proteins by affinity fingerprinting. Chemistry & Biology 2, 107-118.
Tracer purity is critical
It is important to be aware of the propeller effect especially for peptides
and DNA ligands
Labeling position is important. The fluorophore must be as close as possible
to the action.
The Law of Mass Action applies. Become familiar with this Law.
Know the limits of sensitivity for Ligand-Receptor Binding.
Protease Assays:
Bolger R, Checovich W (1994). A new protease activity assay
using fluorescence polarization. Biotechniques 17, 585.
Schade SZ, et al. (1994). Spirochete protease detection by fluorescence
polarization. J Dent Res 73, 248.
Schade SZ, et al. (1995). BODIPY-Alpha-Casein, a pH-Independent protease
substrate for fluorescence polarization (FP) assays. J Dent Res 74, 54.
Kang HC, Haugland RP (1995). Dibenzopynometheneborondifluoride dyes, US
Patent No. 5433896.
Jolley ME (1996). Fluorescence polarization assays for the detection of
proteases and their inhibitors. J Biomolecular Screening 1:1, 33-38.
Non-Specific Tips:
Try FITC-Casein for > pH 6
Try BODIPY-Casein as it is pH independent
Specific Tips:
Use a Fluorescence-Labeled Peptide > 30 residues
Biotin-Peptide-Fluorescein Plus Avidin > 30 residues
BODIPY-Casein Tracer Preparation:
a. BODIPY-FL-C3-SE or BODIPY TR X SE at 2.5 g per mg of Alpha
Casein in 0.1M carbonate buffer, pH 9.0 for 15 minutes at room temperature.
b. Purify through a G25 column in PBS-Azide
c. Aliquot and freeze
FP Protease Assay Protocol
a. Blank (optional)
b. Add tracer to 1 g/ml
c. Add enzyme (may be added before step 1)
d. Incubate
e. Read mP
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