User Manual - TopoGEN, Inc.
Contents
1. Topo Il exists as two isoforms p170 alpha and p180 beta Both isoforms act as type II topoisomerases and will relax superhelical DNA and decatenate kineotoplast DNA kKDNA from the insect trypanosome Crithidia fasciculata The type Il enzymes require MgCL2 ATP and will relax DNA in discrete linking number steps of two This is a prototypical eukaryotic enzyme mechanism that will relax either positively or negative supercoiled plasmids changing DNA linking number in discrete steps of two Because Topo II is important as a decatenase activity in vivo it has an acute preference for binding to DNA nodes or duplex duplex crossovers where it will promote one duplex passage through another intact duplex In this way these enzymes are highly proficient at decatenating interlocked DNA rings This Assay Kit is relatively simple and measures the release of minicircular DNAs by decatenation of an intertangled mass of KDNA Topo II is especially good at performing this reaction in vitro The enzyme binds robustly to KDNA networks and rapidly releases intact 2 5 KB monomeric rings Since kDNA networks are extremely large they fail to enter a 1 agarose gel In contrast the minicircular 2 5 KB rings rapidly migrate into the gel The released decatenated products are somewhat heterogeneous but are predominantly in the form of nicked open circular minicircles and fully closed circular rings Both are considered decatenation products Topo Il2. TopoGEN Copyright TopoGEN Inc 2011 All rights reserved User Manual Topoisomerase Il Assay Kit plasmid based O Catalog Number TG1001 1 100 Reaction Set O Catalog Number TG1001 2 250 Reaction Set Lot Number Shipping and Storage of Reagents The kit may be shipped at ambient temperature or on ice dry ice or wet ice The DNAs should be stored at 4 C and the buffers stored at 20 C upon receipt Avoid frequent freeze thaw cycles with the plasmid as this may contribute to DNA breakage ES matt TopoGEN Inc 108 Aces Alley Port Orange Florida 32128 USA Tel 614 451 5810 Fax 614 559 3932 Orders info topogen com Support support topogen com Website www topogen com Product Application and Disclaimer This product is not licensed or approved for administration to humans or animals It may be used with experimental animals only The product is for in vitro research diagnostic studies only The product in non infectious and non hazardous to human health This information is based on present knowledge and does not constitute a guarantee for any specific product features and shall not establish a legally valid contractual relationship TopoGEN Inc shall not be held liable for product failure due to mishandling and incorrect storage by end user TopoGEN s liability is limited to credit or product replacement Topoisomerase II Assay Kit User Manual l Introduction A Summary Human Topoisomerase II
3. agarose gel results In addition CIA extraction will extract non polar compounds that may interfere with the gel staining with some test drugs Run a 1 agarose until the dye front bromophenol blue is about 4 6 cm down the gel Do not run overnight as this will cause the DNA bands to diffuse Usually a gel gives good separation after 30 min or less we use 4 5V cm Stain with 0 5 ug ml ethidium bromide destain for 15 min in water and photodocument results This is a non ethidium bromide gel separation that works well see gel data below You may also run EB containing gels 0 5ug EB ml with EB in Gel and Buffer beware it is a mutagen so wear gloves at all times do not inhale powder Run gels as above and destain with water for 15 min prior to photo documenting the results see Fig 1 for typical gel result TopoGEN Inc www topogen com Protocol TG1001 4 Version Topoisomerase II Assay Kit User Manual Fig 1 Typical Topo Il reaction products with marker DNAs A 1 EB containing agarose gel was run Lane 1 kDNA Catenated DNA marker Lane 2 KDNA digested with Xho1 Lane 3 KDNA 4 units topo II with 50 uM Etoposide Lane 4 same as lane 3 without etoposide Catenated Se eee kDNA Nicked Open Linear a lt e Circular kDNA a 1 afelaxe D Important Considerations about this kit Marker DNAs are extremely important You should always run decatenated and linear KDNA markers Any nucleas
4. does not induce formation of linear DNA products under the conditions of this assay therefore linear KDNA should not be detected in the gels The Human Topoisomerase Il Assay Kit contains reagents necessary to quantify topo Il activity in crude cell extracts Markers are included to allow unambiguous detection of topo Il even in the presence of contaminating topoisomerase The assay is KDNA based and is highly specific for topo Il Nuclease activity may however cause some degradation of the kKDNA substrate Such degradation will be ATP independent In addition nucleases will generate a smear of degradation products in addition to linear KDNA This kit does not contain enzyme TopoGEN Inc www topogen com Protocol TG1001 2 Version Topoisomerase II Assay Kit User Manual human topoisomerase II is available online by ordering catalog number TG2000H 2 B Kit Contents 100 assay kit size 1 kDNA 20 ug total substrate at the concentration specified on the tube provided Typically one should use 0 1 to 0 2 ug per reaction 2 Decatenated KDNA marker 25 ul in gel loading buffer Run 2 ul of decatenated DNA marker per gel 3 Linearized KDNA marker 25 ul in gel loading buffer run 2 ul of linear marker per gel 4 10x Topo II Incomplete Assay Buffer A 0 5 M Tris HCl pH 8 1 50 M NaCl 100 mM MgCh 5 mM Dithiothreitol 300 ug BSA ml 5 10x ATP Buffer B contains 20mM ATP in water You must mix Buffers A and B together pri
5. e free agarose of reasonable quality can be used from Sigma A positive control with known topo II activity and negative control no extract are also very critical for data interpretation For details on how to make a nuclear salt extract from cells follow our protocol on the website http Awww topogen com p methods protocols html See Methods for Extracting Topoisomerases for Enzyme Activity PDF Alternatively purified human topoisomerase lla is available for purchase TG2000H 2 Agarose gels 1 and running buffers can be any standard nondenaturing electrophoresis buffer example to prepare a 50x of TAE Gel Buffer 242 g Tris base 57 1 ml glacial acetic acid and 100 ml of 0 5 M EDTA Dilute to 1x to use for gel separations EB agarose gels EB at 0 5 ug ml in gel and buffer will improve the resolution of cleavage products nicked open circular KDNA and circular kDNA see Fig 1 above TopoGEN Inc www topogen com Protocol TG1001 5 Version Topoisomerase II Assay Kit User Manual Run gels at a relatively high voltage 100 150 v to facilitate rapid run times Even if the bromophenol blue goes less than a few cm you should have sufficient resolution to detect decatenated kKDNA products After running EB gel should be destained in water or buffer for 15 min prior to photodocumentation E Data Interpretation and additional helpful hints Look carefully at the gels for evidence of kDNA breakdown or degradation ind
6. her questions or comments please feel free to contact us Email info topogen com Telephone 614 451 5810 TopoGEN Inc www topogen com Protocol TG1001 7 Version
7. icating the presence of a nuclease activity usually not a problem Activity can be measured by the disappearance of kKDNA networks catenanes or the formation of decatenated DNA see Fig 1 F Frequently asked questions What are the critical controls Marker DNAs linear kKDNA decatenated and catenated kDNA see Fig 1 are extremely important Include a negative control no extract Be sure to check solvent effects if included or effects of salt used to extract topo from nuclei More than 200mM NaCl from the crude extract will impact negatively on the results What kind of agarose should buy Any nuclease free agarose of reasonable quality from any number of sources can be used Sigma Aldrich works What is the best gel buffer to use Agarose gels 1 and running buffers can be any standard nondenaturing electrophoresis buffer example to prepare a 50x of TAE Gel Buffer 242 g Tris base 57 1 ml glacial acetic acid and 100 ml of 0 5 M EDTA Dilute to 1x to use for gel separations Be sure that the gel also has 1x TAE buffer Why should run EB gels In general Ethidium Bromide EB gels are ideal for testing enzyme activity in this system because it is much easier and faster than the non EB system Be sure to destain with water for 15 min prior to photodocumenting your data If the DNA products are poorly resolved you can simply re electrophorese until resolution improves TopoGEN Inc www topogen com Prot
8. ocol TG1001 6 Version Topoisomerase II Assay Kit User Manual What are the running conditions in terms of time and voltage Run the gels at a relatively high voltage so that the dye front moves 4 5 cm in about 20 30 min Thus the gels run rather fast which accelerates the pace of the assay What reaction volumes do you recommend for these assays Reaction volumes should be 20 30 ul final volume limited by the volume that can be loaded into the wells of the agarose gel The reactions should be assembled on ice in microfuge tubes water buffer and DNA test compound and enzyme which should be added last After adding enzyme the tubes should be transferred to a heating block to initiate the reaction Is proteinase K required Usually it is not necessary however if the nuclear extract is concentrated with protein it may be a good idea to degrade these proteins to improve the cosmetics of your gel I see two decatenated bands in the gel Why is that This is normal when you include EB in the gel system In the presence of EB the minicircles resolve out as nicked ss nick and intact circular Both of these are decatenation products and you can quantify either of these to measure topo II Can you help us with data interpretation Yes we can definitely help The best way to proceed is to send us your data support topogen com with a full description of the experiment We will get back to you quickly with feedback Any furt
9. or to make a 5x Complete Assay Buffer To prepare a fresh stock of the 5x Assay buffer Add equal volumes Buffer A and B example if you need 50 ul of 5x Complete Buffer for a single experiment mix 25 ul of Buffer B with 25 ul of Buffer A The Complete 5x Buffer should be made fresh for each experiment Prepare only the amount as needed each day DO NOT STORE THE 10x COMPLETE ASSAY BUFFER IT IS NOT STABLE 6 5x Stop Buffer gel loading dye 600 ul 5x buffer is 5 Sarkosyl 0 125 bromophenol blue 25 glycerol TopoGEN Inc www topogen com Protocol TG1001 3 Version Topoisomerase II Assay Kit User Manual C Protocol for a typical Reaction Mixture of 20 ul Assemble all reactants in the following order NNN H20 to make up to final volume 20 ul in this case 5x Complete Reaction Buffer made 1 1 of A B 4 ul DNA 1 ul 250 500 ng is idea for most assays Test extract 1 ul vary as needed Incubate 30 minutes at 37 C Stop by addition of 4 ul 5x Stop Buffer Samples may be loaded directly onto the agarose gel at this point Optional Step the samples can be cleaned up by proteinase K digestion 50 ug ml for 15 min at 37 C followed by CIA extraction For CIA extractions add an equal volume 20 ul of Chloroform isoamyl Alcohol or CIA 24 1 vortex briefly spin in a microfuge for 5 sec Withdraw blue colored upper aqueous phase and load onto agarose gel CIA extraction will usually improve the cosmetic quality of the
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