14,15-EET/DHET ELISA kit
Contents
1. from the same sample pool the collected organic phases ethyl acetate together and evaporate under argon gas To cleave the esterified eicosanoids 2 mL of 20 KOH was added and mixed very well The mixture was incubated at 50 C for one hour Prepare a 20 KOH solution from 1 mL 2M KOH and 4 mL methanol final concentration KOH 0 4 N Dilute 2 mL of the aqueous solution with 3 mL of H O Adjust the pH using 20 formic acid to pH 5 Add ethyl acetate 1 part aqueous solution 1 part ethyl acetate vortex thoroughly and centrifuge at 2000 rpm for ten minutes at 22 C Repeat the procedure twice more using an equal volume of ethyl acetate per sample Pool all the organic phase ethyl acetate together and evaporate under argon gas Dissolve the dried residue in a minimal amount of ethanol 20 uL add 20 uL of acetic acid to make a pH of approximately 3 4 In the acidic conditions EET is hydrolyzed to DHET The reaction usually takes 12 h at 45 C or 18 h overnight at room temperature The reaction vial has to be flushed with argon and kept under an argon blanket An argon blanket is like a pouch to keep an argon gas flow during the hydrolysis If an argon blanket is not available at your place you can add clean powdered dry ice to get rid of residual oxygen After reaction add 1 5x water to the sample and extract the sample three times with equal volume of ethyl acetate For each extraction vortex thoroughly and spin down and collect the2. vial of the 14 15 DHET HRP conjugate 0 012 mL with 12 00 mL of 1 X HRP buffer One vial makes enough conjugate for one plate The conjugate must be used the same day and should not be stored for later use Standards Label 5 microtubes as Standard 1 through Standard 5 Dilute the entire contents of Sample Dilution Stock buffer 25 mL with 225 mL deionized water to yield a final volume of 250 mL of 1 X Sample Dilution Buffer Add 0 9 mL of the Sample Dilution Buffer to the microtubes for Standards 1 to 5 Spin down the enclosed 14 15 DHET standard vial 2 uL filled with inert gas and add 1 998 mL of Sample Dilution Buffer to obtain 2 mL of solution Label this Standard 6 Add 0 1 mL of the Standard 6 to the microtube labeled Standard 5 and mix thoroughly Next add 0 1 mL of Standard 5 into the microtube labeled Standard 4 and mix thoroughly Continue to serially dilute the standards using 1 10 dilutions for the remaining standards Samples Samples can be directly diluted into the 1 X Sample Dilution Buffer if it is in solution For extracted and dried samples it is recommended to dissolve the dried up samples with a minimal amount of ethanol of N N dimethyl formanmide DMF 10 uL to 20 uL and vortex well Before ELISA assay add 100 uL of 1 X Sample Dilution Buffer to make the stock sample solution ready for quantification with ELISA The stock sample solution can be further diluted to a proper range of concentration for ELISA test 2727 Second
3. 14 15 EET DHET ELISA kit Catalog Number DH2 DH12 DH22 Store at 20 C FOR RESEARCH USE ONLY V 05262010 Detroit R amp D Inc a Introduction It is well known that arachidonic acid AA will be converted to EET by P4so arachidonic acid epoxygenase AA epoxygenase and EET will be converted to DHET by soluble epoxide hydrolase sEH in vivo Cytochrome P450 2J2 CYP2J2 is a predominant human AA epoxygenase that produces all four EETs In human carcinoma cells rAAV mediated over expression of CYP2J2 resulted in a marked increase in 14 15 DHET level in cell plasma whereas rAAV anti2J2 mediated silence of CYP2J2 expression significantly decreased its production 1 Out EET DHET kit can be used to measure EET levels in cultured cells which express sEH 1 DH2 DH12 or DH22 to be used for 14 15 EET DHET measurement is the same kit as DH1 which is used for the measurement of 14 15 DHET The only difference with DH2 DH12 and DH22 compared with DH1 is the sample preparation step Instructions are provided as to the proper isolation and purification in the following pages Storage and Stability This kit will obtain optimal results if all of the components are stored at the proper temperature prior to use Items should be stored at the designated temperatures upon receipt of this kit All components are stored below 20 C and should not be re frozen and thawed more than necessary Materials Provided Part Item Description Qua
4. 9 EET 0 40 5 s 15 s DiHETE 0 20 11 12 EET 0 05 Arachidonic Acid 0 05 5 6 DHET 0 02 5 6 EET 0 02 Thromboxane B2 0 02 PGE lt 0 01 PGF lt 0 01 6 keto PGF lt 0 01 Recent experiment showed 0 3 cross reactivity Troubleshooting No color present in standard wells The HRP conjugate was not added Redo the assay and add the conjugate at the proper step The HRP conjugate was not incubated for the proper time Redo the assay and incubate for the proper time No color in any wells including the TA wells The TMB substrate was not added Add substrate The TMB substrate was not incubated for the proper time Continue incubation until desired color is reached The color is faint One or all of the incubation times were cut short Redo the assay with the proper incubation times The TMB substrate was not warmed up to room temperature Redo the assay making sure all reagents are at room temperature The lab is too cold Be sure the lab temperature is between 21 27 C and redo the assay The background color is very high The TMB substrate has been contaminated Redo the assay with a fresh bottle of substrate Scattered O D obtained from the sample Redo assay using an 8 channel pipetman making sure that 8 channels are equal volume while loading 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Pag
5. Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 5 of 8 Performing the Assay Plate Setup Each plate must contain a minimum of three blank wells B1 three maximum binding wells Bo and a Six point standard curve S S Each sample should be assayed in triplicate A suggested plate format is shown below weer OO0O0000 0 eeeOOO0O00000 0 IIOOO0O0000 0 IIIO OOO 0000 O JJ90 0000000 IIOOO0O0000 O IIIO OOOO 000 O IIIO OOO 0000 O EE eo SS Samples Standard Dilutions Table Standards Final Concentration pg mL Add Sample Dilution Buffer mL Serial Dilutions Procedure No 6 1 000 000 1 998 2 uL of stock solution No 5 100 000 0 9 Add 0 1 mL of No 6 No 4 10 000 0 9 Add 0 1 mL of No 5 No 3 1 000 0 9 Add 0 1 mL of No 4 No 2 100 0 9 Add 0 1 mL of No 3 No 1 10 0 9 Add 0 1 mL of No 2 Assay Procedure Step 1 Load 200 microliters of Sample Dilution Buffer into the blank B1 wells and 100 microliters of Sample Dilution Buffer into the maximum binding Bo wells Step 2 Load 100 microliters of each of the standards into the appropriate wells Step 3 Load 100 microliters of each of the samples into the appropriate wells Step 4 Load 100 microliters of the diluted 14 15 DHET HRP conjugate in the Bo wells the standard wells and the sample wells Do NOT add HRP conjugate into the By wells Step 5 Incubate the plate at room tem
6. ate to room temperature before proceeding with the assay It is necessary to thoroughly mix the concentrated buffer solutions A stir bar is contained within each buffer solution Sample Preparations EET DHET can be measured after chemically changing EET to DHET However if the EET in cells or in blood is changed to DHET by abundantly expressed soluble epoxide hydrolase measurement of DHET without chemically changing EET to DHET is suitable For example when 14 15 DHET levels were measured in urine samples obtained from Spontaneously Hypertensive rats 14 15 DHET levels in the urine were measured without changing EET to DHET High 14 15 DHET levels were indicative of increased soluble epoxide hydrolase activity of the rat thus soluble epoxide hydrolase dependent hypertension However when P450 2C23 activity of the rat microsomes was measured the rat microsomes were incubated with arachidonic acid substrate of P450 2C23 and then EET DHET levels in the reaction mixture were measured after acid hydrolysis of EET to DHET which was indicative of P450 2C23 activity There are three different protocols which can be used to convert EET into DHET for measurement using the competitive ELISA kit For optimal results please choose the protocol which fits your sample best Protocol 1 EET formation activity measurement 1 Collect and homogenize and or sonicate the cells using a solution containing a final concentration of 0 1 mM TPP triph
7. e 8 of 8 Warranty Detroit R amp D Inc makes no warranty of any kind expressed or implied including but not limited to the warranties of fitness for a particular purpose and merchantability Detroit R amp D Inc DO J Detroit R amp D Inc Metro Center for High Technology Bldg MCHT 2727 Second Ave Suite 4113 Detroit MI 48201 Phone 313 961 1606 Fax 313 963 7130 E mail info detroitrandd com www DetroitRandD com 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com
8. enylphosphine TPP is an antioxidant which looks like precipitate in samples because it does not easily dissolve Before using the stored samples with TPP spin to separate the TPP from the samples 2 Acidify the whole homogenized cells with acetic acid to a pH of approximately 3 4 Measure using standard pH paper Be careful when changing pH by adding 1 uL of acetic acid at a time 3 Extract with ethyl acetate Add an equal volume of ethyl acetate to the homogenized cells and vortex thoroughly Transfer the upper organic phase into a fresh clean tube after centrifugation Then add another equal volume of ethyl acetate to the homogenized cells and repeat the extraction two more times 4 Evaporate the pooled ethyl acetate until all is dried up under argon gas 5 Add 20 uL of ethanol or N N dimethyl formamide DMF to dissolve the dried up residue for reconstitution Add 0 5 mL 1x Sample Dilution Buffer provided in the kit to make a solution Load 100 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com 6 Page 3 of 8 uL into each well in triplicate on the ELISA plate Note We recommend measuring a different dilution of the sample in an attempt to fit the results to the standard curve e g Add 50 uL of the rest of the sample plus 50 uL 1x Sample Dilution Buffer to three wells plus add 10 uL of the rest of the sample plus 90 uL of 1x Sample Dil
9. erage by the maximum binding then multiply by 100 Repeat this formula for the remaining standards 4 Plot the B Bo versus the concentration of 14 15 DHET from the standards using semi log paper 5 Calculate the B Bo for the samples and determine the concentrations utilizing the standard curve 6 Multiply the concentrations obtained for each of the samples by their corresponding dilution factor Typical Results 70 60 gt 50 B 40 NX oo 8 30 20 10 s o EPEE 1 100 10000 1000000 14 15DHET pg mL The data shown here is an example of typical results obtained using the Detroit R amp D 14 15 DHET ELISA kit These results are only a guideline and should not be used to determine values from your samples The user must run their own standard curve every time 0 070 0 869 By wells Bo wells 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 7 of 8 Standard Concentration O D B Bo No 1 10 pg mL 0 540 67 6 No 2 100 pg mL 0 445 55 7 No 3 1 000 pg mL 0 300 37 5 No 4 10 000 pg mL 0 094 11 8 No 5 100 000 pg mL 0 020 2 5 No 6 1 000 000 pg mL 0 011 1 4 Specificity of anti 14 15 DHET IgG The specificity of the 14 15 DHET ELISA was investigated using authentic 14 15 DHET and a panel of eiconsanoids 14 15 DHET 100 00 8 9 DHET 3 30 11 12 DHET 3 30 14 15 EET 1 5 15 s HETE 1 00 8
10. ntity Number 1 14 15 DHET ELISA Solid 96 well plate coated with anti 14 15 DHET antibody in 1 Plate each well 14 15 DHET Standard 2 2 uL Stock standard at a concentration of 1 mg mL 1 14 15 DHET HRP Conjugates 3 2u 1000 X concentrated solution 1 Sample Dilution Buffer s 4 25 mL 10 X solution of Tris buffered saline with preservatives l 5 p a 1 X solution of Tris buffered saline with preservatives 1 Wash Buffer Solution 10 X solution of Tris buffered saline with detergents and 6 25 mL preservatives 1 TMB Substrate 24 mL A solution of TMB tetra methyl benzadine 1 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 2 of 8 Additional Required Materials Not Provided Plate reader with a 450 nm filter An 8 channel adjustable pipetter and an adjustable pipetter Storage bottles Costar cluster tubes 1 2 mL and microcentrifuge tubes Deionized water Precautions Please read all instructions carefully before beginning the assay The reagents in this kit have been tested and formulated to perform optimally This kit may not perform correctly if any of the reagents are replaced or any of the procedures are modified This kit is intended for research use only and is not to be used as a diagnostic Procedural Notes Remove all of the reagents required including the TMB and allow them to equilibr
11. organic phase After three times of extraction pool all the organic phase ethyl acetate together and evaporate under argon 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 4 of 8 7 For ELISA dissolve the sediment in 20 uL of ethanol or DMF vortex thourghly then add 130 uL of 1x Sample Dilution Buffer to make stock solution The stock sample solution can be diluted in a proper range of concentration for ELISA test Check the final pH should be pH 7 4 8 Use the 14 15 DHET ELISA kit to measure DHET which includes DHET converted from EET At the same time measure the DHET level without hydrolysis of EET in the same sample Subtract that value from the EET DHET level and you will obtain the EET level in the sample References Cancer Res 2005 65 4707 15 Circulation 2004 110 2132 Letters in Drug Design amp Discovery 2005 2 239 etc Assay Preparations The solid 96 well plate and TMB solution are provided ready to use The preparations of other assay reagents are detailed below Wash Buffer Mix the solution with a stir bar applying low heat until a clear colorless solution is obtained Dilute the entire contents of the Wash Buffer Concentrate 25 mL with 225 mL of deionized water to yield a final volume of 250 mL of 1 X Wash Buffer This can then be refrigerated for the entire life of the kit HRP Conjugate Dilute 1
12. perature for two hours Step 6 Wash the plate three times with 400 microliters of the diluted Wash Buffer per well 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 6 of 8 Step 7 After the last of the three wash cycles pat the plate dry onto some paper toweling Step 8 Add 200 microliters of the TMB substrate to all of the wells including By wells Step 9 Incubate the plate at room temperature for 15 30 minutes Step 10 Add 50 micoliters of 2 N sulfuric acid to all of the wells Step 11 Read the plate at 450 nm Calculating the Results Most plate readers provide data reduction software that can be used to plot the standard curve and determine the sample concentrations If your plate reader does not have this option then a data reduction program can be used 4 parameter of log log curve fit If you do not have these options the results can be obtained manually as follows 1 Average the absorbance readings from the blanks and subtract that value from each well of the plate to obtain the corrected readings Note Some plate readers do this automatically Consult the user manual of your plate reader 2 Average the corrected absorbance readings from the Bo wells This is your maximum binding 3 Calculate the B Bo for Standard 1 by averaging the corrected absorbance of the two S wells divide the av
13. tract the sample three times with equal volume of ethyl acetate vortex well spin down and collect the organic phase After three times of extraction pool all the organic phase ethyl acetate together and evaporate under argon or nitrogen For ELISA assay dissolve the sediment in 20 uL of ethanol or DMF vortex thourghly then add 130 uL of 1x Sample Dilution Buffer to make stock solution The stock sample solution can be diluted in a proper range of concentration for ELISA test Check the final pH should be pH 7 4 Use the 14 15 DHET ELISA kit to measure DHET which includes DHET converted from EET At the same time measure the DHET level without hydrolysis of EET in the same sample Subtract that value from the EET DHET level and you will obtain the EET level in the sample Protocol 3 Free and esterified EET DHET formation activity measurement 1 Biological samples have to be collected in TPP triphenylphosphine with a final concentration of 0 1 mM TPP is an antioxidant which looks like precipitate in samples because it does not easily dissolve Before using the stored samples with TPP spin to separate the TPP from the samples Acidify the samples with acetic acid to a pH of approximately 3 4 After acidification the samples are extracted three times with ethyl acetate For each extraction add an equal volume of ethyl acetate to the sample vortex thoroughly spin down and collect the organic phase After extracting three times
14. ution Buffer to three wells Perform the ELISA for 14 15 DHET according to the instructions of the manufacturer Protocol 2 Free EET DHET formation activity measurement 1 Biological samples have to be collected in TPP triphenylphosphine with a final concentration of 0 1 mM TPP is an antioxidant which looks like precipitate in samples because it does not easily dissolve Before using the stored samples with TPP spin to separate the TPP from the samples Acidify the samples with acetic acid to a pH of approximately 3 4 After acidification the samples are extracted three times with ethyl acetate For each extraction add an equal volume of ethyl acetate to the sample vortex thoroughly spin down and collect the organic phase After extracting three times from the same sample pool the collected organic phases ethyl acetate and evaporate under argon gas Dissolve the above dried up residue in 20 uL of ethanol then add 20 uL of acetic acid to make the pH approximately 3 4 In the acidic conditions EET is hydrolyzed to DHET The reaction usually takes 12 h at 45 C or 18 h overnight at room temperature The reaction vial has to be flushed with argon and kept under an argon blanket An argon blanket is like a pouch to keep an argon gas flow during the hydrolysis If an argon blanket is not available at your place you can add clean powdered dry ice to get rid of residual oxygen After the reaction add 1 5X water to the sample and ex
Download Pdf Manuals
Related Search