List of Assays
Methodology
Absorbance = Abs, Fluorescence= FI, Fluorescence Polarization = FP, Time resolved Fluorescence = TRF, Fluorescence Resonance Energy Transfer = FRET
Intended Purpose: Determination of kinetic parameters such as Km, Vmax, etc.; IC50; Low, Medium, and High throughput Screening.
Assay
Enzymatische Analysen
Enzymatic assays for:
Kinases (PKC alpha)
TRF
Proteases (HIV1 Protease, Cathepsin B)
FRET, IQF
Small G-proteins; Examples: Protein Gq, Rac
FP
ELISA
Phosphodieasterases (PDE V)Phosphodieasterases (PDE V)
FP
Intended purpose: Ligand, agonist and antagonist binding: Determination of Kd, Bmax, EC50 and IC50 for inhibitors.
Metabolic Enzymes
Abs
ECM (Collagenase, Transglutaminase)
G-protein
FP
Adenylate Cyclase
FAAH
FI
FI
Biliverdin reductase
FP
Estrogen Receptors
FP, TRF
Trimeric G-proteins
Receptors
Receptor activities
hCGbeta, hFc, MCP1, Insulin
Collagen
HRP
FP
Immuno Assays
Intended purpose: establishing assays for protein-protein interactions, preparation of samples, antibodies characterization
RIPA
Cell extractions
Protein detection
SDS-PAGE, WB
Proteomics
Protein quantification
Bradford, Lawry, BCA
FRET-TRF
ELISA
Example II
Example I
Protein-Protein interactions
Phosphorylation & De-Phosphorylationin vitro
According to chosen assay
ELISA, W.B.
Detection of antigens in samples
Ligand protein interaction
Inhibition in binding assays
Competition-based binding assays
Binding assays
IP&W.B., FRET, HTRF, alpha screen, FP
IP&W.B., FRET, HTRF, alpha screen, FP
FRET, HTRF, FP
ELISA, W.B., FACS, IP
Detection of: cytoplasmic, nuclear, membrane, and cytoskeleton proteins
Fluorophore labeling of proteins and peptides
Fluorescein, Biotin
Alexa-fluor
Labeling peptides purification by CRO
Labeling proteins purification on PD10
Western Blotting
Preparing Samples for Western Blotting
Preparing Samples for Quantitative Western Blotting
Tricine SDS Gels
Electrophoretic separation
Electrophoretic Separation for Quantitative Western Blotting
Transfer: iBlot, dry/semi-dry/wet transfer
Quantitative Western Blotting
Developing Western Blots Using the ECL, Chromogenic Methods
Developing Western Blots with Scientific Imaging Film
Visualization of Western Blots on the Kodak Image Station
Abs, Fl, FP, TRF, and more
Compound Characterization
Solubility
Fluorescence Spectroscopy
FP
Abs
3D FI
Chemiluminescence
Proteomic profiling
3D FI
Protein - Peptide/Nucleic acids/Small molecule Interaction
FP, ELISA
FP
Nucleic acids quantification
Biophysical assays
Thermal shift
Protein-small molecule
Other
In-vitro transcription/translation
FRET in confocal microscopy
Cell Based Assays
Cell bank storage available
Growing, freezing, thawing, and packaging different cell lines
Cell extractions and protein detection:
preparing protein whole-cell extracts for quantitative gel analysis
See: Cell Free assays
BL2 facility enable diverse tissue culture activities
Tissue culture methods
Tissue culture methods
Grow and store Cells
Tissue Culture
Methodology
Assay
Potency Assays
Proliferation/Activation/Apoptosis
Example: proliferation of lymph-node cells
BRDU
Toxicity assays
Cell Viability, cytotoxicity, anchorage-dependent cell death, and cellular senescence
Angiogenesis Assay
use ECM matrix gel
WST1, MTT, XTT, Anexcin CY3, Cell titer glo…
Autophagy-a lysosomal degradation pathway for cytoplasmic material and is activated during cellular stress
Cell Adhesion and attachment
Example: Fibroblast attachment assay
Cell Biolabs kits
Two forms of LC3 may be seen by Western blot
Cell migration: Chemotaxis, Haptotaxis, Transmigration, Wound healing
Example: Transwell using mcp1 as chemo-attractent
Matrigel, Transwell + WST1
MTT, XTT, WST1, WST3, IL2 (T cells)
Cell Biolabs kits
CytoSelectâ„¢ ECM Cell Adhesion Assays (Cell Biolabs)
Glucose Uptake in 3T3-L1
Induction of cell differentiation (Such as L1 Pre- Adipocytes to differentiated Adipocytes
Induction methods
Interleukins, TNF, growth factors…
Cell activation & proliferation
Binding to folate receptor in KB cells
Proliferation of fibroblasts (CRL2522) and endothelial cells treated with recombinant human proteins
Tgase activity
Cell activation measured by Ca++ influx
Ectopic expression of Proteins in Bacteria & mamalian cell lines
Cell cycle
Detect cell surface receptors and antigens
Trimeric G protein via forskolin induced cAMP
FACS, ELISA
Propidium Iodide (PI) read in FACS
Transfections, Transformations
ELISA
Cell signaling
Binding Assays
Mammalian two hybrid
IP-WB, ELISA
Protein-Protein interaction
Cell Biolabs kits
Gene expression/quantification
mRNA, RNA, miRNA, siRNA, DNA and RNA-Plex
Immunological assays
Isolation of PBMCs from whole blood
Gravity Sedimentation
Magnetic separation
T Cell enrichment
T Cell Activation/Expansion
CD3+CD28 antibodies
Inflamation assays
Other
Confocal microscopy
Lenti-viral particles
Cell permeability assay
Kinetic of drugs internalization into cells
LC-MS/MS
targeted metabolomic
LC-MS/MS
Specific antibodies
Intended purpose: Ligand, agonist and antagonist binding: Determination of IC50 for inhibitors.
Insulin, Calcitonin
Bio Assays
Specific Assays
Assay / Activity
Methodology
Detection of peptides in serum
Absorbance following HRP
Detection of antibodies in serum
Human Fc from conditioned medium
HRP
ELISA-HRP
Cytokines
Detection of Cytokines in serum
Specific proteins
Penetration of Blood Brain Barrier
Extraction of evans Blue/ Fluorescein and labeled proteins from rat brain
Fluorescence/Absorbance
Insulin, Calcitonin
Absorbance following HRP
Pharmacokinetic analysis
Quantification of small molecules/peptide in biological fluids and tissue
LC-MS/MS
Early in vitro ADMETox
Assay
Methodology
Absorbtion
Intestinal absorbtion
Caco2 cells
Absorption
Intestinal absorption
Caco2 cells
Distribution
Binding to plasma proteins
FP
Metabolism
Metabolic Stability
Microsomes, Hepatocytes
Microsomes
Esterases metabolism
Recombinant CYP450 isozymes
CYP-Inhibition
CYP-Induction
mRNA
Toxicity
Cytotoxicity
WST1-viability assay
Cardiotoxicity
Binding assay-FP