{"product_id":"dna-polymerase-theta-activity-assay-kit-bht20700006","title":"DNA Polymerase Theta Activity Assay Kit","description":"\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBackground\u003c\/h4\u003e\n\u003cp\u003eDNA polymerase theta (Pol θ) is involved in an end-joining pathway of DNA double-strand breaks. Over \nexpression of Pol θ is found in many cancers, including stomach, colon, breast and lung cancers, and \ncorrelated with poorer patient survival. Because suppression of gene expression of Pol θ results in \nsensitivity of cells to ionizing radiation and some DSB-inducing drugs, Pol θ is a validated anti-cancer drug \ntarget. \n\nDescription \n\nThe Aurora DNA Polymerase Theta activity assay kit is a homogeneous fluorescence-based assay for \nscreening inhibitors that block DNA polymerase activity of DNA Pol θ. \n\n The assay is fast, convenient, and requires just two steps. In the first step, the DNA Pol θ enzyme \nsynthesizes double-stranded DNA using a DNA template in the presence of dNTP. In the second step, a dye \nthat binds to double-stranded DNA is added to the solution resulting in the increase of fluorescence, \nintensity of which can be measured with a fluorescent plate reader at the excitation wavelengths of 495 \nnm and emission wavelengths of 525 nm. \n\nMaterials supplied \n\nCatalogue Number \n362201 \n362204 \n4687 \n362003 \n4930 \n362202 \n\nItem \n2X Assay Buffer \n20 µM DNA template \n10 mM dNTP \nRecombinant DNA Pol θ CTD \nDye solution \nStop solution \nBlack low binding 96 well plate \n\nAmount \n25 mL \n7 µL \n5 µL \n5 µL \n15 µL \n3 mL \n1 \n\nStorage \n-20°C \n-20°C \n-20°C \n-80°C \n-20°C \n-20°C \nRT \n\nMaterials Needed but not supplied \n\nA microplate reader capable of measuring fluorescence at excitation wavelengths of 495 nm and \nemission wavelengths of 525 nm. \n\n1. 0.5 M DTT \n2. Adjustable micro-pipettor \n3. Sterile Tips \n\nStability \n\n12 months if stored under the indicated conditions. \n\nAurora Biolabs LLC, San Diego, CA 92121, USA. www.aurorabiolabs.com, \n\n \n1\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eMaterials Not Supplied\u003c\/h4\u003e\n\u003cul\u003e\n\u003cli\u003e0.5 M DTT\u003c\/li\u003e\n\u003cli\u003eAdjustable micro-pipettor\u003c\/li\u003e\n\u003cli\u003eSterile Tips\u003c\/li\u003e\n\u003cli\u003ePrepare 1X buffer containing 1 mM DTT.\u003c\/li\u003e\n\u003cli\u003ePrepare the inhibitor compound solution.\u003c\/li\u003e\n\u003cli\u003ePrepare DNA Pol Theta solution.\u003c\/li\u003e\n\u003cli\u003eAdd the inhibitor solution\u003c\/li\u003e\n\u003cli\u003eIncubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli\u003ePrepare substrate solution\u003c\/li\u003e\n\u003cli\u003eIncubate the plate at 30°C for 1 hour.\u003c\/li\u003e\n\u003cli\u003ePrepare dye solution\u003c\/li\u003e\n\u003cli\u003eIncubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli\u003eMeasure the fluorescent intensity\u003c\/li\u003e\n\u003c\/ul\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Protocol\u003c\/h4\u003e\n\u003col style=\"padding-left:1.2em\"\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 1.\u003c\/strong\u003e Prepare the inhibitor compound solution. If the inhibitor compound is dissolved in water, make a solution of the compound 5-fold higher than the final concentration in 1X assay buffer (since you will add 5 µl to the 25 µl reaction). Then make a series of dilutions in 1X assay buffer from this solution to your preferred concentrations. If the inhibitor compound is dissolved in DMSO, make a 100-fold higher concentration of the compound than the highest concentration you want to test in DMSO. Then make a 20-fold dilution in 1X assay buffer (at this step, the compound concentration is 5-fold higher than the final concentration and the DMSO concentration is 5%). Then make a series of dilutions in 5% of DMSO from this solution to your preferred concentrations. Since 5 µl of the compound solution will be added to the 25 µl reaction, the final concentration of DMSO in all of reactions is 1%.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 2.\u003c\/strong\u003e Prepare DNA Pol Theta solution. Thaw DNA Pol θ CTD enzyme (catalogue number 362003) on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted enzyme at -80°C. Note: DNA Pol θ CTD enzyme is sensitive to freeze\/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted enzyme. Dilute DNA Pol Theta enzyme 650-fold (1:650) in 1X assay buffer with 1 mM DTT. Add 10 µl of diluted enzyme solution to each of positive control well and inhibitor test well. Add 1X buffer to each of background well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 3.\u003c\/strong\u003e Add the inhibitor solution Add 5 µl of 1X assay buffer to each background well and positive control well if the inhibitor is diluted in 1X buffer. Add 5 µl of 1X assay buffer with 5% DMSO to each of background well and positive control well if the inhibitor is diluted in 1X buffer with 10% DMSO. Add 5 µl of diluted inhibitor solution from Step 2 to each of the inhibitor test well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 4.\u003c\/strong\u003e Incubate at room temperature for 30 minutes (optional).\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 5.\u003c\/strong\u003e Prepare substrate solution During the incubation of the enzyme and the inhibitor solution, prepare substrate solution containing 0.125 µM DNA template (dilute from 20 µM DNA template, catalogue number 362004) and 25 µM dNTP (dilute from 10 mM dNTP) in 1X assay buffer. Make only enough solution as need for the assay. Store the remaining 20 µM DNA template and 10 mM dNTP solution to -20°C. Add 10 µl of the substrate solution to each of well, including background wells, positive control wells and the inhibitor test wells.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 6.\u003c\/strong\u003e Incubate the plate at 30°C for 1 hour.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 7.\u003c\/strong\u003e Prepare dye solution Dilute the Dye solution 200-fold in Stop solution (catalogue number 362202). Make only enough solution as need for the assay. Store the remaining Dye solution to -20°C. Add 25 µl the Dye solution to each well.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 8.\u003c\/strong\u003e Incubate at room temperature for 30 minutes.\u003c\/li\u003e\n\u003cli style=\"margin-bottom:6px\"\u003e\n\u003cstrong\u003eStep 9.\u003c\/strong\u003e Measure the fluorescent intensity Measure the fluorescent intensity at the excitation wavelengths of 495 nm and the emission wavelengths of 525 nm. Positive Control Inhibitor Test\u003c\/li\u003e\n\u003c\/ol\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eData Analysis\u003c\/h4\u003e\n\u003cdiv style=\"background:#f8fbfb;border-left:3px solid #1a5c58;padding:10px 14px;margin:8px 0;border-radius:4px\"\u003e\n\u003cstrong\u003eStep 2 — Calculate % Activity\u003c\/strong\u003e\u003cbr\u003e\u003ccode style=\"font-size:0.9em\"\u003e% Activity = (S − N) \/ (P − N) × 100\u003c\/code\u003e\u003cbr\u003e\u003csmall\u003eS = sample signal  |  P = positive control (100%)  |  N = negative control (0%)\u003c\/small\u003e\n\u003c\/div\u003e\n\u003cp\u003eCalculate percentage activity of the enzyme \n\n% Activity= \n\n(Fp – Fb) – (Fi-Fb) \nFp - Fb \n\nX 100 \n\nWhere Fp refers to fluorescent intensity of the positive control, Fb refers to fluorescent intensity of \nbackground, and Fi refers to fluorescent intensity of the inhibitor. Graph the percentage activity as a function of the inhibitor concentration to determine the IC50 of the test \ninhibitor. No CPD refers to no compound control \n(compound vehicle only).\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eAssay Validation\u003c\/h4\u003e\n\u003cdiv style=\"background:#f0f7f6;border:1px solid #c8dada;border-radius:6px;padding:12px 16px;margin:8px 0\"\u003e\n\u003cstrong\u003eAssay Validation Data\u003c\/strong\u003e\u003cbr\u003e\n\u003cspan\u003e\u003cem\u003eValidated IC\u003csub\u003e50\u003c\/sub\u003e:\u003c\/em\u003e \u003cstrong\u003e19 nM\u003c\/strong\u003e\u003c\/span\u003e\u003cbr\u003e\n\u003c\/div\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eBiological Pathway \/ Process\u003c\/h4\u003e\n\u003cp\u003eDNA Double-Strand Break Repair (End-Joining TMEJ)\u003c\/p\u003e\n\u003c\/div\u003e\n\u003cdiv class=\"bhc-assay-section\"\u003e\n\u003ch4\u003eTherapeutic \/ Disease Area\u003c\/h4\u003e\n\u003cp\u003eOncology (POLQ-overexpressing cancers: breast; lung; colon; stomach)\u003c\/p\u003e\n\u003c\/div\u003e","brand":"Aurora Biolabs","offers":[{"title":"96 reactions","offer_id":53238302114157,"sku":"362101-96","price":839.0,"currency_code":"USD","in_stock":false},{"title":"384 reactions","offer_id":53238305554797,"sku":"362101-384","price":1199.0,"currency_code":"USD","in_stock":false}],"url":"https:\/\/www.ebiohippo.com\/products\/dna-polymerase-theta-activity-assay-kit-bht20700006","provider":"BioHippo","version":"1.0","type":"link"}