ab118973 MMP-1 Inhibitor Screening Kit - Abcam

1. OverviewThe Matrix metalloproteinase-1 (MMP-1, Interstitial collagenase,fibroblast collagenase) is a member of a multigene family of calciumdependent,zinc-containing endoproteinases, the matrixmetalloproteinases (MMPs). The MMPs are responsible for thedegradation of the extracellular matrix (ECM) including collagens,elastins, gelatin, matrix glycoproteins and proteoglycan duringnormal development and disease processes. MMPs are regulated byhormones, growth factors and cytokines. MMP-1 belongs to thesubclass, the collagenases, and along with MMP-8, and MMP-13 arethe only members of the MMP family that are capable of degradingthe types I, II and III interstitial collagens with high efficiency. Thesecollagens are primarily found in bone, cartilage and skin.In Abcam’s MMP-1 Inhibitor Screening Kit, MMP-1 hydrolyzes aspecific FRET substrate to release a quenched fluorescent group,which can be detected at Em/Ex = 490/520nm. In presence of potentMMP-1 inhibitors the hydrolyzation of substrate will be inhibited orstopped.The kit provides a rapid, simple, sensitive, and reliable test suitableas a high throughput screening assay of MMP-1 inhibitors. Forcomparison of the relative efficacy of test inhibitors, a controlinhibitor, GM 6001 (IC 50 = 0.4 nM for MMP-1) is included.3

3. Components and StorageA. Kit ComponentsItemQuantityAssay BufferSubstrateMMP-1 Enzyme (Lyophilized)25 mL0.2 mL1 vialInhibitor Control (5 µM GM 6001) 20 µL* Store kit at -20°C, protect from light.• Warm the assay buffer to room temperature before use.• Briefly centrifuge vials before opening.• Read the entire protocol before performing the assay.MMP-1 ENZYME: Reconstitute the MMP-1 enzyme with 220 µlAssay Buffer. Aliquot and store the MMP-1 stock solution at -80°C.Avoid repeated freeze/thaw cycles. Use within one week.5

B. Additional Materials Required• Microcentrifuge• Pipettes and pipette tips• Fluorescent microplate reader• 96-well plate• Orbital shaker6

4. Measurement: Read Ex/Em = 490/520 nm R 1 at T 1 . Read R 2again at T 2 after incubating the reaction at 37°C for 30 min, protectedfrom light.The RFU of fluorescence generated by hydrolyzation of substrate is:∆RFU = R 2 – R 1Note:It is recommended to read kinetically to choose the R 1 and R 2 withinthe linear range.8

5. Data AnalysisSet the ∆RFU of Enzyme Control as the 100 % Relative ActivityValue and calculate the relative activity for each candidate inhibitoras follows:% Relative Activity =∆RFU of Candidate∆RFU of Enzyme Controlx 100%9

GM6001 Inhibits MMP-1 Activity at Different Concentrations10

6. TroubleshootingProblem Reason SolutionAssay notworkingUnexpectedresultsAssay buffer atwrong temperatureProtocol step missedPlate read atincorrect wavelengthUnsuitable microtiterplate for assayMeasured at wrongwavelengthSamples containimpeding substancesUnsuitable sampletypeSample readings areoutside linear rangeAssay buffer must not be chilled- needs to be at RTRe-read and follow the protocolexactlyEnsure you are usingappropriate reader and filtersettings (refer to datasheet)Fluorescence: Black plates(clear bottoms)Luminescence: White platesColorimetry: Clear platesIf critical, datasheet will indicatewhether to use flat- or U-shapedwellsUse appropriate reader and filtersettings described in datasheetTroubleshoot and also considerdeproteinizing samplesUse recommended samplestypes as listed on the datasheetConcentrate/ dilute samples tobe in linear range11

SampleswithinconsistentreadingsLower/Higherreadings insamplesandstandardsUnsuitable sampletypeSamples prepared inthe wrong bufferSamples notdeproteinized (ifindicated ondatasheet)Cell/ tissue samplesnot sufficientlyhomogenizedToo many freezethawcyclesSamples containimpeding substancesSamples are too oldor incorrectly storedNot fully thawed kitcomponentsOut-of-date kit orincorrectly storedreagentsReagents sitting forextended periods oniceIncorrect incubationtime/ temperatureIncorrect amountsusedRefer to datasheet for detailsabout incompatible samplesUse the assay buffer provided(or refer to datasheet forinstructions)Use the 10kDa spin column(ab93349)Increase sonication time/number of strokes with theDounce homogenizerAliquot samples to reduce thenumber of freeze-thaw cyclesTroubleshoot and also considerdeproteinizing samplesUse freshly made samples andstore at recommendedtemperature until useWait for components to thawcompletely and gently mix prioruseAlways check expiry date andstore kit components asrecommended on the datasheetTry to prepare a fresh reactionmix prior to each useRefer to datasheet forrecommended incubation timeand/ or temperatureCheck pipette is calibratedcorrectly (always use smallestvolume pipette that can pipetteentire volume)12

Problem Reason SolutionStandardcurve is notlinearNot fully thawed kitcomponentsPipetting errors whensetting up thestandard curveIncorrect pipettingwhen preparing thereaction mixAir bubbles in wellsConcentration ofstandard stockincorrectErrors in standardcurve calculationsUse of otherreagents than thoseprovided with the kitWait for components to thawcompletely and gently mix prioruseTry not to pipette too smallvolumesAlways prepare a master mixAir bubbles will interfere withreadings; try to avoid producingair bubbles and always removebubbles prior to reading platesRecheck datasheet forrecommended concentrations ofstandard stocksRefer to datasheet and re-checkthe calculationsUse fresh components from thesame kitFor further technical questions please do not hesitate tocontact us by email (technical@abcam.com) or phone (select“contact us” on www.abcam.com for the phone number foryour region).13

UK, EU and ROWEmail: technical@abcam.comTel: +44 (0)1223 696000www.abcam.comUS, Canada and Latin AmericaEmail: us.technical@abcam.comTel: 888-77-ABCAM (22226)www.abcam.comChina and Asia PacificEmail: hk.technical@abcam.comTel: 108008523689 ( 中 國 聯 通 )www.abcam.cnJapanEmail: technical@abcam.co.jpTel: +81-(0)3-6231-0940www.abcam.co.jpCopyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.All information / detail is correct at time of going to print.15