Spec. Sheet - Detroit R&D, Inc.
Contents
1. 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 References 1 Liu H Zhao Y Nie D Shi J Fu L Li Y Yu D and Lu J Association of a Functional Cytochrome P450 4F2 Haplotype with Urinary 20 HETE and Hypertension J Am Soc Nephrol 19 714 721 2008 2 Meseguer et al Kidney Androgen Regulated Protein Transgenic Mice Show Hypertension and Renal Alterations Mediated by Oxidative Stress Circulation 119 1908 1917 2009 3 Dolegowska B Blogowski W Domanski L Is it possible to predict the early post transplant allograft function using 20 HETE measurements A preli2. com www detroitRandD com Page 6 of 9 Typical Results QC on 12 10 08 Net OD at 450 nm of Bo 1 667 30 min color development B B0 LT 20 HETE pg mi The data shown here is an example of typical results obtained using the Detroit R amp D 20 HETE 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 By wells 0 063 Bo wells 1 667 Standard Concentration O D B Bo No 1 10 pg mL 1 630 97 8 No 2 100 pg mL 1 597 95 8 No 3 1 000 pg mL 1 319 79 1 No 4 10 000 pg mL 0 687 41 2 No 5 100 000 pg mL 0 428 25 7 No 6 1 000 000 pg mL 0 356 21 3 Specificity of anti 20 HETE Anti 20 HETE did not cross react with 14 15 and 11 12 DHETs PGE and showed almost no cross reactivity even with structurally extremely similar arachidonic acid linoleic acid and linolenic acid as shown in the competitive ELISA analysis shown below 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 9 120 A o bA 1 400 X o 2HETE a Arachidonic Acid 2 _ Linoleic Acid 7 Linolenic Acid a gt 20HETE pg mL Troubleshooting No color present in standard wells The HRP conjugate was not added Redo the assay and add the conjugate at the proper step
3. 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 Quantity Number 1 20 HETE ELISA Solid 96 well plate coated with anti 20 HETE antibody in each 1 Plate well 2 20 HETE Standard 1 2 uL Stock standard at a concentration of 1 mg mL 20 HETE HRP 3 Conjugates 1 12 uL 1000 X concentrated solution Sample Dilution 4 Buffer 10 X solution of Tris buffered saline with preservatives 1 25 mL 5 e 1 X solution of Tris buffered saline with preservatives 1 6 Wash Buffer Solution 10 X solution of Tris buffered saline with detergents and 1 25 mL preservatives TMB Substrate i 7 24 mL A solution of TMB tetra methyl benzadine 1 Rev 09 17 2012 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 2 of9 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 in
4. 20 HETE ELISA kit Catalog Number 20H1 20H11 20H21 20H101 Store at 20 C FOR RESEARCH USE ONLY Detroit R amp D Inc a Introduction This competitive ELISA kit is for determination of 20 HETE also known as 20 OH AA levels in biological samples The specificity of the 20 HETE ELISA was investigated using authentic 20 HETE and a panel of fatty acids which based on their structure might be anticipated to compete with 20 HETE for binding to antibodies for 20 HETE Anti 20 HETE did not cross react with 14 15 and 11 12 DHETs PGE and showed almost no cross reactivity even with structurally extremely similar arachidonic acid AA linoleic acid and linolenic acid as shown in the competitive ELISA analysis Considering the only difference between 20 HETE and AA is an oxygen molecule the specificity of the Detroit R amp D 20 HETE ELISA is a surprise Human essential and salt sensitive hypertensions were related to differential AA metabolism by cytochrome P450 CYP 4A which has AA w hydroxylase 20 HETE synthesis activity Increased circulating insulin inhibits 20 HETE synthesis in obese hypertensive subjects Recently CYP4F2 genetic variants which increased urinary 20 HETE secretion were found to be correlated with the risk for hypertension in a Chinese population This kit can be used for the determination of 20 HETE in serum plasma cells and tissues following proper isolation and purification Instructions are provided as to
5. 299000000000 999000000000 EE EM 20 ES L Samples 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 5 of 9 Standard Dilutions Table No No No No No 6 c9 4 3 2 2 fil Standards Final Concentration pg mL Add Sample Dilution Buffer mL Serial Dilutions Procedure No 1 000 000 1 998 2 uL of stock solution 100 000 0 9 Add 0 1 mL of No 6 10 000 0 9 Add 0 1 mL of No 5 1 000 0 9 Add 0 1 mL of No 4 100 0 9 Add 0 1 mL of No 3 10 0 9 Add 0 1 mL of No 2 Assay Procedure Step 1 Load 200 microliters of Sample Dilution Buffer into the blank B 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 20 HETE 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 temperature for two hours Step 6 Wash the plate three times with 400 microliters of the diluted Wash Buffer per well 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
6. 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 1 to 5 Spin down the enclosed 20 HETE 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 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 000660660600 000000000 299000000000 929900000000 O 299000000000 299000000000
7. ail 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. d Dilute 1 4 e g 80 uL sample 320 uL 1x Sample Dilution Buffer Check the final pH should be pH 7 4 When calculating the concentration consider the dilution factor In this case 150 uL total sample volume from 1 8 mL plasma 12 fold concentration and then 80 sample in 400 uL SDB 5 fold dilution Since the samples are concentrated 2 4 fold to get the actual concentration you must divide by 2 4 Perform the ELISA for 20 HETE according to the instructions of the manufacturer 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 9 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 vial of the 20 HETE 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
9. ethanol so that the final conc of KOH 0 4 N Vortex and incubate for 1 h at 50 C Add 1 5X H 0 to the solution and adjust pH with 20 formic acid to pH S Re extract the solution with ethyl acetate 1 part aqueous solution 1 part ethyl acetate and dry 20 HETE measurement in tissues l 2 3 4 Homogenize g of tissue 4 mL of H2O and 0 01 mg TPP Acidify the homogenate by adding 8 uL of acetic acid to each homogenate Extract with an equal amount of ethyl acetate vortex thoroughly spin down and collect the organic phase Repeat this extraction twice more and combine all of the organic phases Dry the organic phase with argon or nitrogen gas Saponification if needed see 20 HETE measurement in cells Dissolve the dried residue from above step 4 with N N dimethyl formamide DMF Add approximately 20 uL of DMF to reconstitute the dried up residue Dilute further with 1x Sample Dilution Buffer Add approximately 0 5 mL of 1x Sample Dilution Buffer and centrifuge at 10 000 rpm for five minutes at room temperature The supernatant will be used for ELISA Perform the ELISA for 20 HETE according to the instructions of the manufacturer 20 HETE measurement in plasma or serum 1 Combine 1 8 mL of plasma adjusted with approximately 20 uL of acetic acid to pH 4 and 1 8 mL of ethyl acetate Vortex thoroughly Centrifuge at 2000 rpm for ten minutes at 22 C Three phases should result i Upper organic phase et
10. hyl acetate phase lipoproteins ii Interphase proteins iii Lower phase aqueous phase Collect the upper organic phase a and set aside Discard the interphase Transfer the lower phase with a glass pipette to a new tube and repeat the ethyl acetate extraction step 2 more times Evaporation of pooled organic phase There should be approximately 5 6 mL of the ethyl acetate phase a Dry the pooled organic phase in a Speedvac to get the extracted sediment b Saponification to cleave fatty acid from glycerol backbone Dissolve the dried up residues b in 2 mL of 20 KOH solution for preparation see 20 HETE in cells Vortex thoroughly and incubate for 1 hat 50 C This will yield an aqueous solution c Dilute 2 mL of the aqueous solution c with 3 mL of H O Adjust the pH using 20 formic acid 132 uL to pH 5 5 Add ethyl acetate 1 part aqueous solution c 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 Collect the upper phase with saponified lipids Dry the pooled ethyl acetate upper phase d in a Speedvac yielding the dried sample sediment e Store the sediment e at 20 C For ELISA assay dissolve the dried sample residue e in 20 uL N N dimethyl formamide DMF then add 130 uL of 1x Sample Dilution Buffer For the competitive 20 HETE ELISA the above 150 uL sample needs to be further dilute
11. ker discovery Biochem Anal Biochem 1 5 2012 18 Fordsmann JC Ko RW Choi HB Thomsen K Witgen BM Mathiesen C Lonstrup M Piilgaard H MacVicar BA Lauritzen M Increased 20 HETE synthesis explains reduced cerebral blood flow but not impaired neurovascular coupling after cortical spreading depression in rat cerebral cortex J Neurosci 33 2562 2570 2013 19 Stobart JL Lu L Anderson HD Mori H Anderson CM Astrocyte induced cortical vasodilation is mediated by D serine and endothelial nitric oxide synthase PNAS USA 110 3149 3154 2013 20 Tunctan 20 HETE mimetics or inhibitors in the treatment of cancer patients with sepsis and septic shock International Journal of Cancer Studies amp Research IJCR 2 101 2013 21 Tunctan et al NS 398 reverses hypotension in endotoxemic rats Contribution of eicosanoids NO and peroxynitrite Prostaglandins amp other Lipid Mediators 104 105 93 108 2013 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 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 9 of 9 Detroit R amp D Inc a 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 m
12. minary report Transpl Int 2009 22 546 553 4 Liu et al Overexpression of cytochrome P450 4F2 in mice increases 20 hydroxyeicosatetraenoic acid production and arterial blood pressure Kidney International 75 1288 1296 2009 5 Wang et al Selective Inhibitors of CYP2J2 Related to Terfenadine Exhibit Activity Strongly against Human Cancers in vitro and in vivo JPET 152017 2009 6 Cuez Malik Tunctan et al A synthetic analogue of 20 HETE 5 14 HEDGE reverses endotoxin Induced hypotension via increased 20 HETE levels associated with decreased iNOS protein expression and vasodilator prostanoid production in rats Basic Clin Pharmacol Toxicol 106 378 388 2010 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 8 of 9 7 Malik et al 2 3 4 5 Tetramethoxystilbene prevents deoxycorticosterone salt induced hypertension contribution of cytochrome P 450 1B1 Am J Physiol Heart Circ Physiol 299 H1891 H1901 2010 8 Malik et al Cytochrome P450 1B1 contributes to angiotensin II induced hypertension and associated pathophysiology Hypertension 56 667 2010 9 Tunctan et al Contribution of vasoactive eicosanoids and nitric oxide production to the effect of selective cyclooxygenase 2 inhibitor NS 398 on endotoxin induced hypotension in rats Basic Clin Pharmacol Toxicol 107 877 882 2010 10 Imaizumi et al L 4F differentially alter
13. qual volume of ethyl acetate to the homogenized cells and vortex very well Place the upper organic phase into a fresh clean tube after centrifugation Then add another equal volume of ethyl acetate to the homogenized cells to start the second time extraction It is strongly recommended that extraction is performed three times 4 Evaporate the pooled ethyl acetate from the extractions until all has dried up under argon or nitrogen gas 5 Saponification if needed see below 6 Add 20 uL N N dimethyl formamide DMF to dissolve the dried up residue for reconstitution Add 0 5 mL of 1x Sample Dilution Buffer provided in kit to make a solution Load 100 uL in each well in triplicates on the ELISA plate Note We recommend measuring a different dilution of sample in attempt to fit the results to the standard curve e g load 3 wells with 50 uL of the rest of sample plus 50 uL of 1x Sample Dilution Buffer and 3 wells with 10 uL of the rest of sample plus 90 uL of 1x Sample Dilution Buffer 7 Perform the ELISA for 20 HETE according to the instructions of the manufacturer 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd com www detroitRandD com Page 3 of9 Saponification to cleave fatty acid from glycerol backbone 1 Dissolve dried fatty acids obtained from 3X ethyl acetate extractions in 2 mL of 20 KOH solution make working solution 1 mL of 2 M KOH 4 mL m
14. s plasma levels of oxidized fatty acids resulting in more anti inflammatory HDL in mice Drug Metab Letters 4 139 148 2010 11 Cervenka Kramer Falck Imig Hammock et al Combined inhibition of 20 HETE formation and of EET degradation attenuates hypertension and hypertension induced end organ damage in Ren 2 transgenic rats Clinical Science 118 617 632 2010 12 Cervenka Kramer Falck Imig et al Intrarenal CYP 450 metabolites of arachidonic acid in the regulation of the nonclipped kidney function in two kidney one clip Goldblatt hypertensive rats J Hypertens 28 582 593 2010 13 Hu Wang et al Peripheral and central augmentation indexes in relation to the CYP4F2 polymorphisms in Chinese J Hypertens 29 501 508 2011 14 Buharalioglu et al Piroxicam reverses endotoxin induced hypotension in rats contribution of vasoactive eicosanoids and nitric oxide Basic Clin Pharmacol Toxicol 186 194 2011 15 Na Bangchang et al Study on the association between environmental cadmium exposure cytochrome P450 mediated 20 HETE heme oxygenase 1 polymorphism and hypertension in Thai population residing in a malaria endemic areas with cadmium pollution Environ Toxicol Pharmacol 31 416 426 2011 16 Onoe et al Increase of 20 HETE synthase after brain ischemia in rats revealed by PET study with 11 C labeled 20 HETE synthase specific inhibitor J Cereb Blood Flow Metab 32 1737 1746 2012 17 Dong H Metabolomic profiling of lipids for biomar
15. structions 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 equilibrate 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 There are different protocols for isolating and purifying 20 HETE depending on the medium in which it is in Listed below are the different protocols For optimal results follow the appropriate protocol based on the biological sample present 20 HETE measurement in cells expressing Cytochrome P459 4A 1 Collect and homogenize and or sonicate the cells using a solution containing a final concentration of 0 1 mM TPP triphenylphosphine 0 03 0 05 mg mL TPP is an antioxidant which looks like a precipitate in samples because it does not easily dissolve Before using the stored samples containing TPP spin samples to separate the precipitated TPP from sample solution 2 Acidify the whole homogenized cells with acetic acid to a pH of approximately 3 4 Measure using standard pH paper 3 Extraction with ethyl acetate Add an e
16. 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 Average the corrected absorbance readings from the Bo wells This is your maximum binding Calculate the B Bo for Standard 1 by averaging the corrected absorbance of the two S wells divide the average by the maximum binding then multiply by 100 Repeat this formula for the remaining standards Plot the B Bo versus the concentration of 20 HETE from the standards using semi log paper Calculate the B Bo for the samples and determine the concentrations utilizing the standard curve Multiply the concentrations obtained for each of the samples by their corresponding dilution factor 2727 Second Ave Suite 4113 Detroit MI 48201 tel 313 961 1606 fax 313 963 7130 info detroitrandd
Download Pdf Manuals
Related Search