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HIV-1 Integrase Assay Kit
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1. Reaction buffer 1x contains both manganese and magnesium and requires the addition of 0 4 ul of BME mL of reaction buffer before use Prepare 2 ml of reaction buffer for each well Reaction buffer is stable for one week stored at 2 8 C after addition of BME Add BME to the appropriate amount of buffer do not activate the entire 220 ml bottle unless all will be used The reaction buffer may settle in the bottle and it should be mixed swirled gently before use DS and TS DNAs The DS and TS DNAs are provided as 100X solutions that should be diluted 100 fold into reaction buffer before use The diluted 1X DS and TS DNA solutions are not stable and the amount required for an experiment should be prepared just before use The 100X stocks of these DNAs are virtually unaffected by multiple freeze thaw cycles The TS DNA is light sensitive and may leech onto polypropylene tubes HIV 1 Integrase Protein Stability The HIV 1 integrase protein should be stored at 20 C or colder and is stable to freeze thaw cycles with 10 12 possible Before each use the enzyme solution should be thawed on ice for five minutes and then re frozen Plate Washing Steps Washing can be done manually with a wash bottle or with an automatic plate washer The liquid should be completely removed from the plate wells after each washing step by patting the plate down onto a stack of paper towels Replicates We recommend all samples and controls are
2. Blocking Buffer EZ 1702 30 mL DS Oligo DNA 100X Solution EZ 1703 120 uL HIV 1 Integrase 2 uM EZ 1704 60 uL Sodium Azide 20 EZ 1705 2X1mL TS Oligo DNA 100X Solution EZ 1706 60 uL HRP Antibody Solution EZ 1707 12 mL Dilution Plate EZ 1708 1 plate Streptavidin coated 96 well Plate EZ 1709 1 plate Wash Buffer Concentrate 20X 82710 60 mL TMB Peroxidase Substrate XB 1006 12 mL Stop Solution XB 1007 12 mL Instruction Manual NA 1 TECHNICAL ASSISTANCE Please refer any technical questions to info xpressbio com SAFETY INFORMATION Sodium azide may react with lead and copper plumbing to form explosive azide compounds When disposing of reagents flush with copious quantities of water The MSDS for this kit is available online at www expressbiotech com STORAGE CONDITIONS The streptavidin coated plate reaction buffer block buffer sodium azide HRP antibody TMB wash solution and the stop solution should be placed at 2 8 C The DS and TS DNAs and the integrase enzyme should be stored at 20 C or cooler The kit is stable for one year under these conditions Microwell strips of the streptavidin coated plate that are not used after opening the foil pouch should be returned to the pouch along with the sachet of desiccant closed with a pouch sealer or adhesive tape and stored at 2 8 C until the expiration date on the label Reaction buffer 2 ml per reaction well should be activated by the addition of B mercaptomethanol B
3. For Research Use Only Not for Diagnostic Use HIV 1 Integrase Assay Kit Version 3 0 Catalog Number EZ 1700 INTRODUCTION HIV DNA is integrated into host DNA by a viral encoded integrase and this enzyme activity is a likely target site in the HIV life cycle to block viral infection The XpressBio HIV 1 Integrase Assay Kit is a non radioactive assay used to quantitatively measure integrase activity the effects of interacting proteins anti viral compounds and other test articles on HIV 1 integrase activity Streptavidin coated 96 well plates are coated with a double stranded HIV 1 LTR U5 donor substrate DS DNA containing an end labeled biotin Full length recombinant HIV 1 integrase protein is loaded onto the DS DNA substrate Integrase test articles are added to the enzyme reaction and then a different double stranded target substrate TS DNA containing a 3 end modification is added to the reaction mixture The HIV 1 integrase cleaves the terminal two bases from the exposed 3 end of the HIV 1 LTR DS DNA and then catalyzes a strand transfer recombination reaction to integrate the DS DNA into the TS DNA The products of the reaction are detected colorimetrically using an HRP labeled antibody directed against the TS 3 end modification Sodium azide is included in the kit as a positive control compound that inhibits HIV 1 integrase catalytic activity KIT CONTENTS Product Catalogue Per Kit Reaction Buffer EZ 1701 230 mL
4. ME 0 4 wl 1 ml just prior to assay BME activated reaction buffer is stable for one week at 2 8 C Reaction buffer may settle during storage so the bottle should be warmed to 37 C and mixed before use REAGENTS AND EQUIPMENT SUPPLIED BY THE USER e Pipettors and sterile tips e Disposable gloves e 14 5 M B mercaptoethanol BME e Paper towels e Sterile distilled deionized water e A 37 C incubator e A 37 C water bath e A 96 well plate reader capable of reading at 450 nm NOTES BEFORE STARTING General Comments Carefully review the protocol before beginning since small deviations may lead to discrepancies in the final results All incubation steps should be performed within 2 min of the indicated times Each lot of ExpressBio HIV Integrase Assay Kit has been extensively tested and the conditions under which the kit is shipped and stored have been shown empirically to not impact assay performance As with all other 96 well applications there may be a slight difference in assay performance when the plate outer wells and especially the plate corners are used Consequently the inner wells of the plate should be used preferentially in your experimental design whenever critical results are required Wash Buffer Concentrate Wash buffer is provided as a 20X concentrate Mix 50 mL of 20X wash buffer concentrate with 950 mL sterile distilled water to make a 1X wash buffer solution before use Reaction Buffer
5. NA Thaw the HIV 1 integrase on wet ice or at 2 8 C 5 min before it is needed and centrifuge the tube briefly ex 10 000 RPM X 5 sec before use Dilute the enzyme 1 300 into reaction buffer 2 uL HIV 1 integrase and 598 uL reaction buffer Aspirate the liquid from the plate wells and wash three times with 200 ul reaction buffer Add 100 uL of reaction buffer negative control or integrase enzyme solution positive control per well and incubate for 30 min in a 37 C incubator Include reaction buffer only replicates without integrase as a no enzyme negative control Return the reaction buffer to the 37 C water bath 5 Add Inhibitors or Test Articles Prepare test articles by diluting to 2X final desired test concentration in reaction buffer For example prepare a 20 uM or 20 pg mL solution and serially dilute in reaction buffer when a 10 uM or 10 pg mL high test concentration is desired for the assay Azide solution diluted to 0 30 2X concentration or 0 15 final 1X concentration inhibits approximately 50 of the integrase activity Include 50 uL reaction buffer negative and positive control replicates no test article in each experiment Test articles may contain up to 10 dimethyl sulfoxide DMSO since the integrase reaction is only marginally affected by the presence of up to 5 DMSO in the final reaction Aspirate the liquid from the plate wells and wash them three times with 200 uL reaction buffer Add 50 uL per we
6. d 100 ul of the assay reaction wells to 100 ul of dH2O into the dilution plate strip or well EZ 1709 provided do not use the SA coated plate strips and read the absorbance at 450 nM The absorbance values from the diluted samples should be multiplied by 2 DATA ANALYSIS AND ASSAY PERFORMANCE Experimental data are analyzed as described below typically duplicate or triplicate determinations are obtained for each control sample drug test article in each experiment Determine the mean blank absorbance reaction buffer negative control in the assay from the no integrase no test article control usually less than 0 25 OD units and subtract this background absorbance from the other readings Calculate the mean standard deviation SD and CV SD mean X 100 for the background corrected absorbance of integrase alone positive control and test articles replicate wells Convert the data to percent control activity by dividing the mean absorbance of test articles by that of the integrase alone control and multiplying by 100 The mean absorbance of the test articles divided by the mean integrase control activity multiplied by the associated CV provides the percent adjusted standard deviation The CV SD for the 100 integrase alone control Typical assay results for buffer no integrase integrase alone no azide and integrase plus azide each point run in triplicate are tabulated below for the inhibition of the HIV 1 integrase when
7. d 5 4 respectively The data from a typical experiment with the HIV 1 integrase inhibitor Elvitegravir EVG are tabulated below Mean OD Sid Sample ID Corrected Deviation CV Control Corrected Activity Reaction 0 000 0 007 Eau aa a ee Alone EVG EVG EVG EVG EVG EVG EVG EVG A graph illustrating the dosage dependent inhibitory effect of Elvitegravir and Raltegravir on the catalytic activity of HIV 1 integrase is shown below 120 Percent Integrase Activity 0 2 S 0 0 005 0 010 0 025 0 050 0 075 0 100 0 250 0 500 1 00 3 00 5 00 Drug Concentration uM E Elvitegravir Raltegravir The inhibitory concentration of Elvitegravir and Raltegravir that reduces integrase activity by 50 ICs59 and 90 ICg0 may be interpolated from the data and the curve In this experiment Elvitegravir showed an IC5o of 40 nM and an ICoo of 975 nM while Raltegravir showed an ICs of 175 nM and an ICgo of 2 88 uM TROUBLESHOOTING GUIDE Suggestion x Dilute stopped reaction 1 1 in dH2O into a blank strip or well do not use SA coated plate Dilute integrase enzyme at 1 350 in reaction buffer see step 4 above Problem Integrase alone signal gt 3 0 Integrase alone signal lt 0 5 Spin down integrase before use Dilute integrase enzyme 1 250 in step 4 above Increase the TMB incubation time to 20 30 min Wells are stained blue Add stop reage
8. ll of each test article in reaction buffer reaction buffer alone for positive and negative controls and incubate for 5 min at room temperature 6 Add TS DNA Dilute the required amount of TS DNA 100X solution 100 fold in reaction buffer 10 uL TS DNA 100X solution and 990 uL reaction buffer per mL Add 50 uL of the 1X TS DNA solution per well directly to the 50 uL buffer test articles already present in the wells Mix the reactions by tapping the plate gently against a stationary hand 3 5 times Incubate for 30 min at 37 C Detection of Reaction Products 7 Add HRP Antibody Aspirate the liquid from the plate wells and wash five times with 300 uL wash solution Add 100 uL HRP antibody solution per well and incubate for 30 min at 37 C 8 Add TMB Peroxidase Substrate Aspirate the liquid from the plate wells and wash five times with 300 uL wash solution Add 100 uL TMB peroxidase substrate solution per well and incubate for 10 minutes at room temperature 9 Add TMB Stop Solution Add 100 uL TMB stop solution directly to the wells containing the TMB substrate Burst any large bubbles by using a pipette tip Read the absorbance of the wells for a minimum of 0 1 sec using a plate reader set at 450 nm Plates should be read within 10 min of adding TMB stop solution If the OD 450 nM absorbance is above the range of the plate reader the reactions can be dilute in order to realize a more accurate reading or end point Ad
9. nt before reading plates or read plate at 405 nM instead of 450 nM Background is high gt 0 35 Replace reaction buffer Use reaction buffer within one week of adding BME Swirl mix the reaction buffer bottle before use Review plate washing steps above EXPERIENCED USERS PROTOCOL 1 Prewarm reagents 100 uL DS oligo 30 min at 37 C 2 5X 300 uL wash buffer 200 uL block 30 min at 37 C 3 3 X 200 uL reaction buffer wash 100 uL of 1 300 dilution of integrase in reaction buffer 30 min at 37 C 4 3 X 200 uL reaction buffer wash 50 ul reaction buffer or test article in reaction buffer 5 min at room temperature 5 50 uL of TS oligo 30 min at 37 C 6 5 X 300 uL wash buffer 100 uL HRP antibody 30 min at 37 C 7 5X 300 uL wash buffer 100 uL well of TMB substrate 10 min at room temperature 8 100 uL TMB stop solution read OD at 450 nM CONTRACT RESEARCH Need a hand with your research Would you like independent confirmation of your results Why not let us perform the HIV 1 integrase assay for you Contact us for more information CONTACT INFORMATION XpressBio Express Biotech International 503 Gateway Drive West Thurmont MD 21788 USA Toll free 888 562 8914 www XpressBio com info XpressBio com Tel 301 228 2444 Fax 301 560 6570
10. tested using 2 3 replicate wells in each experiment The use of additional replicates for the blank no integrase and the integrase alone controls no test article may reduce variability in the assay HIV INTEGRASE ASSAY KIT PROTOCOL DS Coating and Blocking of SA Plate 1 Prewarm reagents Place the reaction buffer and the blocking solution in a 37 C water bath for 10 min before starting the assay Prewarm all the other components of the kit except the HIV 1 integrase enzyme by placing them at room temperature 2 Coat with DS DNA Remove any strip wells from the streptavidin coated 96 well plate that will not be used and reseal them in the foil pouch containing desiccant Dilute the required amount of DS DNA 100X solution 100 fold in reaction buffer 10 uL DS DNA 100X solution and 990 uL reaction buffer Add 100 uL of 1X DS DNA solution per well and incubate for 30 min in a 37 C incubator Return the reaction buffer to the 37 C water bath 3 Blocking the plate Aspirate the liquid from the plate wells and wash five times with 300 uL 1x wash buffer Add 200 uL of blocking buffer per well and incubate for 30 min in a 37 C incubator If required the plate may now be placed at 2 8 C overnight and later placed in a 37 C incubator for 20 min before continuing with the protocol however the kit performs slightly better if the entire protocol is performed in one day Integrase Reaction 4 Load the integrase onto DS D
11. treated with azide Mean OD er Sample ID Corrected Deviation CV Control Corrected Activity 100 0 5 1 8 3 5 8 0 24 42 71 95 A graph illustrating the dosage dependent inhibitory effect of azide on the catalytic activity of the HIV 1 integrase is shown below Azide Blocks the Catalytic Activity of Wild type HIV 1 Integrase Relative Percent Integrase Activity o 0 05 0 15 0 25 0 45 0 60 Percent Azide Concentration 0 75 1 0 2 0 Wild type HIV 1 Integrase The inhibitory concentration of azide that reduces integrase activity by 50 ICs59 may be interpolated from the curve manually or using more sophisticated programming In this example azide showed an ICs5o of 0 15 and an ICoo of 0 57 A summary of the absorbance values observed for background corrected integrase alone integrase and for the ICs and IC values for sodium azide observed in three independently performed experiments are shown below Expt IntegraseAlone ICs NaN3 IC YNaN3 1 2 9 0 08 0 152 0 568 2 2 8 0 16 0 143 0 537 3 2 6 0 05 0 124 0 613 All 2 77 0 12 0 140 0 012 0 573 0 031 Mean SD background corrected absorbance of six integrase alone control wells Note The absorbance values generated by integrase alone may vary between experiments but the ICs and ICgp values are reproducible with CVs 8 5 an
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