OxiSelect™ 8-iso-Prostaglandin F2α ELISA Kit
Contents
1. Table 1 Preparation of 8 iso PGF2a Standard Curve Note Do not store diluted 8 iso PGF2a Standard solutions Assay Protocol Note Each 8 iso PGF2a Standard and unknown samples should be assayed in duplicate or triplicate A freshly prepared standard curve should be used each time the assay is performed 1 Add 100 uL of the diluted Anti 8 iso PGF2a Antibody to the Goat Anti Rabbit Antibody Coated Plate Incubate 1 hour at 25 C on an orbital shaker 2 Remove the antibody solution from the wells Wash wells 5 times with 300 uL 1X Wash Buffer per well After the last wash empty the wells and tap microwell plate on absorbent pad or paper towel to remove excess wash solution Note Thorough washing is necessary to remove all of the azide present in the antibody solution 3 Combine 55 uL of the 8 iso PGF2a standard or sample and 55 uL of 8 iso PGF2a HRP conjugate in a microtube and mix thoroughly Transfer 100 uL of the combined solution per well A well containing Sample Diluent can be used as a control Incubate 1 hour at 25 C on an orbital shaker AN 5 CELL BIOLABS INC JE a 4 Remove the combined solution from the wells Wash 5 times with 300 uL of 1X Wash Buffer per well After the last wash empty wells and tap microwell plate on absorbent pad or paper towel to remove excess wash solution 5 Add 100 uL of Substrate Solution to each well Incubate at room temperature for 10 30 minutes on an orbital shaker2. Product Manual OxiSelect 8 iso Prostaglandin F2a ELISA Kit Trial Size Catalog Numbers STA 337 T 32 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Lipid peroxidation is a well defined mechanism of cellular damage in animals and plants Lipid peroxides are unstable indicators of oxidative stress in cells that decompose to form more complex and reactive compounds such as isoprostanes The isoprostanes are a type of eicosanoids produced non enzymatically through the oxygen radical induced peroxidation of tissue phospholipids and lipoproteins Isoprostanes are prostaglandin like compounds that appear in normal plasma and urine samples but are elevated by oxidative stress in tissue plasma and urine 8 iso Prostaglandin F2a also known as 8 epi PGF2a 8 isoprostane or 15 isoprostane F2t is an isoprostane that has been shown to be useful for the assessment of oxidative stress in vivo It is produced in membrane phospholipids from non cyclooxygenase and cyclooxygenase peroxidation pathways derived from arachidonic acid 8 iso Prostaglandin F2a 8 iso PGF2qa is a potent vasoconstrictor a mutagen in 3T3 cells as well as vascular smooth muscle cells and also a possible pathophysiological mediator that can alter membrane integrity It has been implicated in atherogenesis and elevated levels are associated with hepatorenal syndrome rheuma
3. 12 000 rpm in a microcentrifuge The clear supernatant can be used in the assay or stored at 20 C or below for future use Before assaying check to be sure each neutralized sample is in the pH range of 6 8 If it is not adjust the pH to this range by adding 100 uL of the sample to 100 uL of the provided Neutralization Solution e Urine samples Acid hydrolysis of urine samples is necessary to break the bonds which hold lipid and non lipid components together prior to ELISA Urine sample is acidified to pH 3 0 by adding 4 h g CELL BIOLABS INC vA a 1 10 volume of 1N HCl Example Add 100 uL of IN HCl to 1 mL of urine sample Acidified urine sample should be further diluted in PBS or Sample Diluent 1 4 to 1 8 before ELISA Preparation of 8 iso PGF2a Standards 1 Prepare fresh standards by diluting the 8 iso PGF2a Standard from 200ug mL to 0 2 ug mL in Sample Diluent for a 1 1000 final dilution Example Add 5 uL of 8 iso PGF2a Standard stock tube to 4 995 mL of Sample Diluent 2 Prepare a series of the remaining 8 iso PGF2a standards according to Table 1 Standard 8 iso PGF2a Standard Sample Diluent 8 iso PGF2a Standard Tubes uL uL pg mL 1 5 uL of Standard Stock 4995 uL 200 000 2 250 uL of Tube 1 750 uL 50 000 3 250 uL of Tube 2 750 uL 12 500 4 250 uL of Tube 3 750 uL 3 125 5 250 uL of Tube 4 750 uL 781 6 250 uL of Tube 5 750 uL 195 7 250 uL of Tube 6 750 uL 49 8 0 uL 200 uL 0
4. 4172 2167 1095 1000184 Pereira S et al 2015 Effect of N acetyl L cysteine on insulin resistance caused by prolonged FFA elevation J Endocrinol doi 10 1530 JOE 14 0676 Zabala V et al 2015 Potential contributions of the tobacco nicotine derived nitrosamine ketone NNK in the pathogenesis of steatohepatitis in a chronic plus binge rat model of alcoholic liver disease Alcohol Alcohol doi http dx doi org 10 1093 alcalc agu083 Macedo R C S et al 2015 Effects of chronic resveratrol supplementation in military firefighters undergo a physical fitness test a placebo controlled double blind study Chem Biol Interact 227 89 95 Tom E N et al 2014 Treatment with an extract of Terminalia superba Engler amp Diels decreases blood pressure and improves endothelial function in spontaneously hypertensive rats J Ethnopharmacol 151 372 379 King A L et al 2014 Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase nitric oxide dependent Proc Natl Acad Sci U S A 111 3182 3187 Gajendragadkar P R et al 2014 Effects of oral lycopene supplementation on vascular function in patients with cardiovascular disease and healthy volunteers a randomised controlled trial PLoS One 9 e99070 Fofonka A et al 2014 Effects of vildagliptin compared with glibenclamide on glucose variability after a submaximal exercise test in patients with type 2 diabetes study protocol for a randomized controll
5. 6 Stop the enzyme reaction by adding 100 uL of Stop Solution to each well Results should be read immediately color will fade over time 7 Read absorbance of each well on a microplate reader using 450 nm as the primary wave length Example of Results The following figures demonstrate typical 8 iso PGF2a results One should use the data below for reference only This data should not be used to interpret actual results 2 5 1 5 OD 450nm 0 5 0 T T T T T 1 10 100 1000 10000 100000 1000000 8 isoprostane pg mL Figure 1 8 iso PGF2a ELISA Standard Curve AN _ CELL BIOLABS INC yA a E O Q O Negative Diluted Diluted Diluted Diluted Diluted Diluted Control 1 2 1 4 1 8 1 16 1 32 1 64 Human Urine Dilutions Figure 2 Dilutions of Human Urine tested with 8 iso PGF2a ELISA Cross reactivity of 8 iso Prostaglandin F2a ELISA Kit Compounds Cross Reactivity 8 iso PGF2a 100 PGFla 4 6 PGF2a 1 85 PGE1 0 19 TXB2 0 023 PGB1 0 02 PGE3 0 012 6 keto PGFla 0 008 13 14 dihydro 15 keto PGF2a 0 008 6 15 keto 13 14 dihydro PGFla 0 005 8 iso PGE1 lt 0 001 PGA2 lt 0 001 PGJ2 lt 0 001 7 AS ELL BIOL for Life ccs References 1 2 3 4 5 Banerjee M Kang K H Morrow J D et al 1992 Am J Physiol 263 H660 H663 Morrow J D Hill K E Burk R F et al 1990 Proc Natl Acad Sci USA 87 9383 9387 Morrow J D Harris
6. T M Roberts L J 1990 Anal Biochem 184 1 10 Vacchiano C A and Tempel G E 1994 J Appl Physiol 77 2912 2917 Wang Z Ciabattoni G Cre minon C et al 1995 Pharmacol Exp Ther 275 94 100 Recent Product Citations 1 10 11 12 13 Tong M et al 2015 Differential contributions of alcohol and the nicotine derived nitrosamine ketone NNK to insulin and insulin like growth factor resistance in the adolescent rat brain Alcohol Alcohol doi 10 1093 alcalc agv 101 Tassone E J et al 2015 Low dose of acetylsalicylic acid and oxidative stress mediated endothelial dysfunction in diabetes a short term evaluation Acta Diabetol 52 249 256 Alway S E et al 2015 Green tea extract attenuates muscle loss and improves muscle function during disuse but fails to improve muscle recovery following unloading in aged rats J Appl Physiol 1985 118 319 330 Sagun G et al 2015 Levels of F2 isoprostane in Behcet s disease Correlation with cardiometabolic risk factors Redox Rep doi 10 1179 1351000215Y 0000000008 Paneni F et al 2015 Adverse epigenetic signatures by histone methyltransferase set7 contribute to vascular dysfunction in patients with type 2 diabetes mellitus Circ Cardiovasc Genet 8 150 158 Dallatu M K et al 2015 The role of hypoxia inducible factor prolyl hydroxylation pathway in deoxycorticosterone acetate salt hypertension in the rat J Hypertens doi 10
7. be of anti 8 iso PGF2a rabbit IgG Sample Diluent Part No 233702 T One 20 mL bottle Neutralization Solution Part No 233705 T One 10 mL bottle 10X Wash Buffer Part No 310806 T One 30 mL bottle Substrate Solution Part No 310807 T One 4 mL amber bottle Stop Solution Part No 310808 T One 4 mL bottle 8 iso PGF2a Standard Part No 233703 T One 10 uL tube of 200 ug mL 8 iso PGF2o in DMSO 9 8 iso PGF2a HRP Conjugate Part No 233704 T One 25 uL tube of 8 iso PGF2a0 HRP conjugate Oe NE eh Materials Not Supplied 1 Protein samples such as purified protein plasma serum cell lysate 2 Deionized water 3 5 uL to 1000 uL adjustable single channel precision micropipettes with disposable tips 4 50 uL to 300 uL adjustable multichannel micropipette with disposable tips 3 JaN CELL BIOLABS INC A a Bottles flasks and conical or microtubes necessary for reagent preparation Reagents and materials necessary for sample extraction and purification Multichannel micropipette reservoir Plate orbital shaker or rotator O oo oS oN p Microplate reader capable of reading at 450 nm 620 nm as optional reference wave length Storage Upon receipt store the Anti 8 iso PGF2a Antibody 8 iso PGF2a HRP Conjugate and 8 iso PGF2a Standard at 20 C Make aliquots as necessary to avoid freeze thaw cycles Store all other kit components at 4 C Any partial or unused components should return to their proper stora
8. ed trial DIABEX VILDA Trials 15 424 Zhang J et al 2014 Sodium ferulate inhibits neointimal hyperplasia in rat balloon injury model PLoS One 9 e87561 CELL BIOLABS INC Z a Bine 14 15 16 17 18 19 Zis P et al 2014 Memory decline in Down syndrome and its relationship to iPF2alpha a urinary marker of oxidative stress PLoS One 9 e97709 Dugas T R et al 2014 Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase nitric oxide dependent PNAS 111 3182 3187 Karakus E et al 2013 Agomelatine an antidepressant with new potent hepatoprotective effects on paracetamol induced liver damage in rats Human and Experimental Toxicology 10 01177 0960327112472994 Mollo R et al 2012 Effect of a lipoic acid on platelet reactivity in type 1 diabetic patients Diabetes Care 35 196 197 Thompson C M et al 2012 Comparison of the effects of hexavalent chromium in the alimentary canal of F344 rats and B6C3F1 mice following exposure in drinking water implications for carcinogenic modes of action Toxicol Sci 125 79 90 Ungvari Z et al 2011 Age associated vascular oxidative stress Nrf2 dysfunction and NF KB activation in the nonhuman primate Macaca mulatta J Gerontol A Biol Sci Med Sci 10 1093 gerona glr092 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their ins
9. ge temperatures Safety Considerations e Some kit components contain azide which can react with copper or lead piping Flush with large volumes of water when disposing of reagents e Some kit reagents are caustic or hazardous and should be handled accordingly Preparation of Reagents e 1X Wash Buffer Dilute the 10X Wash Buffer to 1X with deionized water Stir to homogeneity e Anti 8 iso PGF2a Antibody Immediately before use dilute the Anti 8 iso PGF2a Antibody 1 1000 with Sample Diluent e 8 iso PGF2a HRP Conjugate Immediately before use dilute the conjugate 1 80 with Sample Diluent Only prepare enough of the diluted conjugate for the number of wells immediately used e Substrate Solution Prior to use warm the Substrate Solution to room temperature Note Do not store diluted Anti 8 iso PGF2a Antibody 8 iso PGF2a HRP Conjugate or 8 iso PGF 2a Standard solutions Preparation of Samples Hydrolysis of lipoprotein or phospholipid coupled 8 iso Prostaglandin F2a 8 iso PGF2 a is required to measure both free and esterified isoprostane To hydrolyze this ester bond the sample is usually treated with 2N NaOH at 45 C for 2 hours e Serum plasma tissue lysate samples Use 1 part of 10N NaOH for every 4 parts of liquid sample After incubation at 45 C for 2 hours add 100 uL of concentrated 10N HCI per 500 uL of hydrolyzed sample The sample could turn milky after this addition Centrifuge the samples for 5 minutes at
10. jugate compete for binding to the antibody bound to the plate After this brief incubation and wash a substrate to the HRP is added The HRP activity results in color development that is directly proportional to the amount of 8 iso PGF2a conjugate bound to the plate and inversely proportional to 2 AN CELL BIOLABS INC S a the amount of free 8 iso PGF2a in the samples or standards The 8 iso PGF2a content in an unknown sample is determined by comparing with the known predetermined standard curve Please read the complete kit insert prior to performing the assay Related Products STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation STA 325 OxiSelect Oxidative RNA Damage ELISA Kit 8 OHG Quantitation STA 330 OxiSelect TBARS Assay Kit MDA Quantitation STA 331 OxiSelect MDA Immunoblot Kit STA 344 OxiSelect Hydrogen Peroxide Peroxidase Assay Kit STA 347 OxiSelect In Vitro ROS RNS Assay Kit Green Fluorescence STA 816 OxiSelect N epsilon Carboxymethyl Lysine CML Competitive ELISA Kit STA 817 OxiSelect Advanced Glycation End Products AGE Competitive ELISA Kit STA 832 OxiSelect MDA Competitive ELISA Kit 10 STA 838 OxiSelect HNE Adduct Competitive ELISA Kit SOn Or ED Pe EE BN SS Kit Components 1 Goat_Anti Rabbit Antibody Coated Plate Part No 250001 T One strip well microplate containing 32 wells 8 x 4 Anti 8 iso PGF2a Antibody Part No 233701 T One 10 uL tu
11. toid arthritis carcinogenesis as well as atherosclerosis 8 iso PGF2a circulates in the plasma and is excreted in the urine 8 iso PGF 2a circulates as an esterified LDL Phospholipid and as a free acid Normal human plasma and urine 8 iso PGF2a is about 40 100 pg mL and about 190 pg mg of creatinine respectively Methods for determining total 8 iso PGF2a usually require alkaline hydrolysis of 8 iso PGF2a esters from tissues followed by extractions phase separations and thin layer chromatography 8 iso Prostaglandin F2a 8 iso PGF2a The OxiSelect 8 iso Prostaglandin F2a ELISA Kit is an enzyme immunoassay developed for rapid detection and quantification of 8 iso PGF2a The quantity of 8 iso PGF2a in samples is determined by comparing its absorbance with that of a known 8 iso PGF2a standard curve Each Trial Size 8 iso Prostaglandin F2a ELISA Kit provides sufficient reagents to perform up to 32 assays including the standard curve and unknown samples Assay Principle Cell Biolabs 8 iso PGF2a kit is a competitive enzyme linked immunoassay ELISA for determining levels of 8 iso PGF2a in a variety of biological samples such as plasma urine serum or tissue extracts An antibody to 8 iso PGF2a is incubated in pre coated microtiter plate wells Upon washing 8 iso PGF2qa standards or treated samples are mixed with an 8 iso PGF2a HRP conjugate and added simultaneously to the wells The unconjugated or free 8 iso PGF2a and 8 iso PGF2a HRP con
12. tructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2013 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing JAA 9 CELL BIOLABS INC
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