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

Enzymatic Assays

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 

CytoSelect™ 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

Contact

smart@assays.co.il

 

Tel: +972 (0)77-300-1087

Mobil: +972 (0)54-7241-251

Location

3 Pinchas Sapir St.,

Weizmann Science Park

Ness Ziona,

7414003 ISRAEL

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