Name: direct measurement of kdm1a target engagement using chemoprobe-based immunoassays
Uploaded: 2019-06-13T13:00:00
Description: Watch this Scientific Journal Video about Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays at JoVE.com

This study was financed by Oryzon Genomics. S.A., Hoffman-La Roche, and partially supported by the CIIP-20152001 and RETOS collaboration program RTC-2015-3332-1.

Blood samples were obtained from the Instituto de Investigaci&#243;n Biom&#233;dica Sant Pau Biobank according to Spanish legislation (Real Decreto de Biobancos 1716/2011) and approval of the local ethics committees. Studies with animal tissues were performed in accordance with the institutional guidelines for the care and use of laboratory animals (European Communities Council Directive 86/609/EEC) established by the Ethical Committee for Animal Experimentation at the PRAAL-PCB.
1. Pre...

<ol>
	<li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1.">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119 (7), 941-953 (2004).</li><li>Maiques-Diaz, A., Somervaille, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSD1:+biologic+roles+and+therapeutic+targeting.">LSD1: biologic roles and therapeutic targeting.</a> Epigenomics. 8 (8), 1103-1116 (2016).</li><li>Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORY-1001,+a+Potent+and+Selective+Covalent+KDM1A+Inhibitor,+for+the+Treatment+of+Acute+Leukemia.">ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia.</a> Cancer Cell. 33 (3), 495-511 (2018).</li><li>Sugino, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+LSD1+inhibitor+NCD38+ameliorates+MDS-related+leukemia+with+complex+karyotype+by+attenuating+leukemia+programs+via+activating+super-enhancers.">A novel LSD1 inhibitor NCD38 ameliorates MDS-related leukemia with complex karyotype by attenuating leukemia programs via activating super-enhancers.</a> Leukemia. 31 (11), 2303-2314 (2017).</li><li>Kleppe, M., Shank, K., Efthymia, P., Riehnhoff, H., Levine, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysine-Specific+Histone+Demethylase,+LSD1,+(KDM1A)+As+a+Novel+Therapeutic+Target+in+Myeloproliferative+Neoplasms.">Lysine-Specific Histone Demethylase, LSD1, (KDM1A) As a Novel Therapeutic Target in Myeloproliferative Neoplasms.</a> Blood. 126, 601 (2015).</li><li>Jutzi, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LSD1+Inhibition+Prolongs+Survival+in+Mouse+Models+of+MPN+by+Selectively+Targeting+the+Disease+Clone.">LSD1 Inhibition Prolongs Survival in Mouse Models of MPN by Selectively Targeting the Disease Clone.</a> HemaSphere. 2 (3), 54 (2018).</li><li>Mohammad, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Hypomethylation+Signature+Predicts+Antitumor+Activity+of+LSD1+Inhibitors+in+SCLC.">DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC.</a> Cancer Cell. 28 (1), 57-69 (2015).</li><li>Rivers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RN-1,+a+potent+and+selective+lysine-specific+demethylase+1+inhibitor,+increases+&#947;-globin+expression,+F+reticulocytes,+and+F+cells+in+a+sickle+cell+disease+mouse+model.">RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases &#947;-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.</a> Experimental Hematology. 43 (7), 546-553 (2015).</li><li>Rivers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+the+LSD1+inhibitor+ORY-3001+increases+fetal+hemoglobin+in+sickle+cell+mice+and+baboons.">Oral administration of the LSD1 inhibitor ORY-3001 increases fetal hemoglobin in sickle cell mice and baboons.</a> Experimental Hematology. 67, 60-64 (2018).</li><li>Buesa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+LSD1/MAO-B+inhibitor+ORY-2001+prevents+the+development+of+the+memory+deficit+in+samp8+mice+through+induction+of+neuronal+plasticity+and+reduction+of+neuroinflammation.">The dual LSD1/MAO-B inhibitor ORY-2001 prevents the development of the memory deficit in samp8 mice through induction of neuronal plasticity and reduction of neuroinflammation.</a> Alzheimer&#8217;s &amp; Dementia. 11 (7), P905 (2015).</li><li>Schmidt, D. M., McCafferty, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-2-Phenylcyclopropylamine+is+a+mechanism-based+inactivator+of+the+histone+demethylase+LSD1.">Trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1.</a> Biochemistry. 46 (14), 4408-4416 (2007).</li><li>Forneris, F., Binda, C., Vanoni, M. A., Battaglioli, E., Mattevi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+histone+demethylase+LSD1+reads+the+histone+code.">Human histone demethylase LSD1 reads the histone code.</a> Journal of Biological Chemistry. 280 (50), 41360-41365 (2005).</li><li>Gonz#225;lez, E. C., Maes, T., Crusat, C. M., Mu#241;oz, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oryzon+Genomics,+Methods+to+determine+kdm1a+target+engagement+and+chemoprobes+useful+therefor.">Oryzon Genomics, Methods to determine kdm1a target engagement and chemoprobes useful therefor.</a> , (2016).</li><li>Mascaró, C., Ortega, A., Carceller, E., Rruiz Rodriguez, R., Cicero, F., Lunardi, S., Yu, L., Hilbert, M., Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoprobe-based+assays+of+histone+lysine+demethylase+1A+target+occupation+enable+in+vivo+pharmacokinetics+and+-dynamics+studies+of+KDM1A+inhibitors.">Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and -dynamics studies of KDM1A inhibitors.</a> Journal of Biological Chemistry. , (2019).</li><li>Rodriguez-Suarez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Homogeneous+Nonradioactive+Methyltransferase+and+Demethylase+Assays+Targeting+Histone+H3+Lysine+4.">Development of Homogeneous Nonradioactive Methyltransferase and Demethylase Assays Targeting Histone H3 Lysine 4.</a> Journal of Biomolecular Screening. 17 (1), 49-58 (2011).</li><li>Lynch, J. T., Cockerill, M. J., Hitchin, J. R., Wiseman, D. H., Somervaille, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD86+expression+as+a+surrogate+cellular+biomarker+for+pharmacological+inhibition+of+the+histone+demethylase+lysine-specific+demethylase+1.">CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.</a> Analytical Biochemistry. 442 (1), 104-106 (2013).</li><li>Schulz-Fincke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+studies+on+N-Substituted+tranylcypromine+derivatives+lead+to+selective+inhibitors+of+lysine+specific+demethylase+1+(LSD1)+and+potent+inducers+of+leukemic+cell+differentiation.">Structure-activity studies on N-Substituted tranylcypromine derivatives lead to selective inhibitors of lysine specific demethylase 1 (LSD1) and potent inducers of leukemic cell differentiation.</a> European Journal of Medicinal Chemistry. 144, 52-67 (2018).</li><li>Ishii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CETSA+quantitatively+verifies+in+vivo+target+engagement+of+novel+RIPK1+inhibitors+in+various+biospecimens.">CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens.</a> Scientific Report. 7, 13000 (2017).</li><li>Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORY-2001:+An+Epigenetic+drug+for+the+treatment+of+cognition+defects+in+Alzheimer&#8217;s+disease+and+other+neurodegenerative+disorders.">ORY-2001: An Epigenetic drug for the treatment of cognition defects in Alzheimer&#8217;s disease and other neurodegenerative disorders.</a> Alzheimer&#8217;s &amp; Dementia. 12 (7), P1192 (2017).</li></ol>

The protocol presented here was developed to directly measure KDM1A target engagement using a novel KDM1A chemoprobe capture based ELISA. The method has been validated on cultured human cell lines and ex vivo samples from human, rat and mouse and baboon (including PBMCs, lung, brain, skin, tumors), but can be readily applied to other species in which the KDM1A antibody target epitopes and catalytic center are conserved. As OG-881 is an activity based chemoprobe, the sample quality is important and proper manipulation and...

Lysine specific demethylase 1 (KDM1A)1 is a demethylase involved in the control of gene transcription. This protein has emerged as a candidate pharmacological target2 in oncology; including Acute Myeloid Leukemia3 (AML), Myelodysplasia Syndrome (MDS)4, Myelofibrosis (MF)5,6, Small Cell Lung Cancer (SCLC)7; in Sickle Cell Disease (SCD)8,9, and in central nervous system diseases including Alzheimer&#39;s Disease (AD), Multiple Sclerosis (MS); and in aggression10.
Most of the KDM1A inhibiting compounds in clinical development are cyclopropylamine derivatives and inhibit the protein by covalent binding to its flavin adenine dinucleotide (FAD) cofactor11. Inhibition of KDM1A induces gene expression changes, but these changes vary enormously across tissues, cell types, or disease cases. Inhibition of KDM1A also changes histone marks12, yet these changes are generally produced locally at a specific site in the genome, and are again, highly tissue and cell specific.
The protocol was developed to directly measure KDM1A target engagement in biological samples and has been optimized for the use with cyclopropylamine derived inhibitors. The assay is based on ELISA technology and analyzes, in parallel, Total and Free (i.e. unbound by inhibitor) KDM1A in a native protein extract from a biological sample in a solid phase assay. As a first step, the biological sample is lysed in the presence of the biotinylated KDM1A selective chemoprobe OG-88113,14, derived from the selective KDM1A inhibitor ORY-1001 (iadademstat), a potent inhibitor of KDM1A in clinical development for the treatment of oncological disease. The chemoprobe has an IC50 for KDM1A of 120 nM and includes a FAD binding moiety linked to a biotinylated polyethylene glycol (PEG)-tail. The chemoprobe exclusively binds to the free KDM1A, but not to the inhibitor-bound KDM1A in the sample. After the chemoprobe binding, the KDM1A containing complexes in the sample are captured on microtiter plates with streptavidin coated surface to determine free KDM1A, or on plates coated with a monoclonal anti-KDM1A capture antibody to determine total KDM1A. After washing, both plates are incubated with an anti-KDM1A detection antibody, washed again, and incubated with a secondary HRP-conjugated donkey anti-rabbit IgG antibody for detection using a luminescent substrate and quantification by measuring relative light units (RLU) in a luminometer (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59390/59390fig1.jpg" /> 
Figure 1. Schema of ELISA Enzyme linked chemoprobe immunoabsorbent assay for KDM1A target engagement: A) Determination of total KDM1A using sandwich ELISA and B) Determination of free KDM1A using chemoprobe ELISA. <a href="https://www.jove.com/files/ftp_upload/59390/59390fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A standard curve is included in both ELISA plates to verify the linearity of each assay. The determination of KDM1A target engagement in each sample is then calculated as a relative value to the pre-dose or vehicle treated sample.

<table>
	<tbody>
		<tr>
			<td>0,05% Trypsin-EDTA (1X)</td>
			<td>Thermo Scientific</td>
			<td>#25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X Protease Inhibitor Tablets</td>
			<td>Roche</td>
			<td>#11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep well storage block</td>
			<td>VWR</td>
			<td>#734-1679</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well ELISA plates</td>
			<td>Nunc</td>
			<td>#436110</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive black Film</td>
			<td>Perkin Elmer</td>
			<td>#6050173</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive transparent Film</td>
			<td>VWR</td>
			<td>#60941-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated KDM1A probe OG-881</td>
			<td>Oryzon Genomics S.A.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td># 3117057001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Standard</td>
			<td>Thermo Scientific</td>
			<td>#23208)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Protein Assay</td>
			<td>BioRad</td>
			<td>#500-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lysis buffer 10X</td>
			<td>Cell Signaling</td>
			<td>#9803</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 96- well plates</td>
			<td>Hettich</td>
			<td>Rotina 420R</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Thermo Scientific</td>
			<td>#156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Full length, enzymatically active human Recombinant LSD1 / KDM1A</td>
			<td>Active Motif</td>
			<td>#31426</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism 5 Project</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol-Enhacer and Peroxide Solution (Chemiluminescent Substrate)</td>
			<td>Thermo Scientific</td>
			<td>#37074</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader Infinite 200-Tecan</td>
			<td>Tecan</td>
			<td>Infinite 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal capture antibody Anti-KDM1A (N-terminal epitope)</td>
			<td>Abcam</td>
			<td>#ab53269</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle G18 gauge blunt</td>
			<td>BD</td>
			<td>#303129</td>
			<td></td>
		</tr>
		<tr>
			<td>ORY-1001 (iadademstat)</td>
			<td>Oryzon Genomics S.A.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMC separation tubes 10 ml</td>
			<td>Greiner bio-one</td>
			<td>#163288</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMC separation tubes 50 ml</td>
			<td>Greiner bio-one</td>
			<td>#227288</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>#D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate shaker</td>
			<td>Heidolph Instruments</td>
			<td>Rotamax 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma</td>
			<td>#P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal detection antibody Anti-KDM1A (C-terminal epitope)</td>
			<td>Cell Signaling</td>
			<td>#672184BF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody Peroxidase-conjugated Donkey Anti-rabbit IgG</td>
			<td>Thermo Scientific</td>
			<td>#31458</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer cuvette 1.5</td>
			<td>Deltalab</td>
			<td>#302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer for cuvette</td>
			<td>GE Healthcare</td>
			<td>GeneQuant 1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Promega</td>
			<td>#Z704A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>#303172</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 ultrapure water</td>
			<td>Millipore</td>
			<td>Milli-Q Advantage A10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>VWR</td>
			<td>USC200T</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct measurement of kdm1a target engagement using chemoprobe-based immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

The Linearity of Total and Free KDM1A determination.
A Standard Series was prepared as described in step 5.3.2., using 0 to 2500 pg of full-length human recombinant KDM1A enzyme. The RLU values of Total and Free rKDM1A were assessed to verify the linearity (Figure 2A and 2B). Data are represented as mean from 3 experiments with 3 technical replicates (n) &#177; SD. ...

Watch this Scientific Journal Video about Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays at JoVE.com

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Cortisol Measurement in Koala Phascolarctos cinereus Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...