OxiSelect™ Protein Carbonyl Fluorometric Assay
Contents
1. Product Manual OxiSelect Protein Carbonyl Fluorometric Assay Catalog Number STA 307 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS IN Introduction Protein oxidation is defined as the covalent modification of a protein induced either directly by reactive oxygen species ROS or indirectly by reaction with secondary by products of oxidative stress Oxidative modification of proteins can be induced in vitro by a wide array of pro oxidant agents and occurs in vivo during aging and in certain disease conditions There are numerous types of protein oxidative modification The most common products of protein oxidation in biological samples are the protein carbonyl derivatives of Pro Arg Lys and Thr These derivatives are chemically stable and serve as markers of oxidative stress for most types of ROS Traditionally carbonyls have been measured spectrophotometrically through derivitization of the carbonyl group with dinitrophenylhydrazine although this method has limited sensitivity In response to this a new fluorescent compound has been developed that binds specifically to carbonyls of oxidized proteins at 1 1 molar ratio Upon binding with the protein carbonyl the fluorescence produced is a direct measure of protein carbonyls and is more sensitive than the traditional spectrophotometric assay using DNPH Cell Biolabs OxiSelect Protein Carbonyl Fluorometric Assay provide2. Protein Carbonyl Fluorophore 100X Part No 230701 One 50 uL amber vial SX TCA Solution Part No 231502 One 50 mL bottle 3 Sample Diluent 10X Part No 230702 One 20 mL bottle Protein Solubilization Solution Part No 231504 One 15 mL bottle Assay Diluent 4X Part No 234505 One 50 mL bottle aur Fluorophore Standard Part No 230703 One 20 uL amber vial of a 20 mM solution in DMSO Materials Not Supplied 1 1 to 10 mg mL protein samples such as plasma serum cell lysate purified protein Protein assay such as Pierce BCA or Bradford assay p Acetone Streptomycin sulfate SL to 1000 pL adjustable single channel precision micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Bottles flasks and conical or microcentrifuge tubes necessary for reagent preparation Centrifugal filter or concentrator Pern ans Reagents and materials necessary for sample extraction and purification 10 96 well black or fluorescence microtiter plate 11 Fluorescent microplate reader capable of reading 480 nm excitation and 530 nm emission Storage Upon receipt aliquot and store the Protein Carbonyl Fluorophore and Fluorophore Standard at 20 C to avoid multiple freeze thaw cycles Store all other components at 4 C until their expiration dates Preparation of Reagents e 1X TCA Solution Dilute the 5X TCA Solution to 1X with deionized water Sti
3. bonyls To remove nuclei acid add streptomycin sulfate or PEI to a final concentration of 1 and 0 5 respectively incubate samples 30 minutes at room temperature and remove the nuclei acid precipitates by centrifuging at 6000 g for 10 minutes at 4 C Test supernatant accordingly Cells or Tissues Centrifuge cells at 1 500 x g for 5 minutes at 4 C Remove media and resuspend in 1 2 mL cold 1X Sample Diluent Homogenize or sonicate cells or tissues on ice Centrifuge at 10 000 x g for 5 minutes at 4 C Remove supernatant and store on ice Store on ice or at 80 C for long term storage Prior to use adjust the total protein concentration to 1 10 mg mL with 1X Sample Diluent e Serum Collect blood without using an anticoagulant Allow blood to clot for 30 minutes at room temperature Centrifuge blood at 2 000 x g for 15 minutes at 4 C Carefully remove the top yellow serum layer without disturbing the white buffy layer Store on ice or at 80 C for long term storage Prior to use adjust the total protein concentration to 1 10 mg mL with 1X Sample Diluent e Plasma Collect blood using an anticoagulant such as citrate or heparin Centrifuge blood at 1000 x g for 5 minutes at 4 C Carefully remove the top yellow serum layer without disturbing the white buffy layer Store on ice or at 80 C for long term storage Prior to use adjust the total protein concentration to 1 10 mg mL with 1X Sample Diluent e Urine Urine normally has very low
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5. ondria caused by mtDNA toxicity leads to heart failure Am J Physiol Heart Cire Physiol 309 H434 H449 Sadowska Bartosz I amp Bartosz G 2015 Ascorbic acid and protein glycation in vitro Chem Biol Interact doi 10 1016 j cbi 2015 07 006 3 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 hitp dx doi org 10 1093 alcalc agu083 4 Rey B et al 2014 Thyroid status affects membranes susceptibility to free radicals and oxidative balance in skeletal muscle of Muscovy ducklings Cairina moschata J Exp Zool A Ecol Genet Physiol 321 415 421 5 Tong M et al 2014 Therapeutic reversal of chronic alcohol related steatohepatitis with the ceramide inhibitor myriocin Int J Exp Pathol 95 49 63 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions 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 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 in
6. otected from light 4 Add 400 uL of 1X TCA solution to each tube Vortex thoroughly and incubate on ice for 10 minutes 5 Centrifuge the tubes at 10 000 x g for 10 minutes Remove and discard the supernatant 6 Add 1 mL of acetone to each tube and vortex thoroughly in order to break up the pellet If the pellet does not disintegrate upon vortexing use a spatula or other small implement to manually break up the pellet 7 Centrifuge the tubes at 10 000 x g for 10 minutes 8 Repeat the acetone wash two additional times 9 After the last wash remove and discard the supernatant Leave sample tube lids open allow the sample pellet to dry out thoroughly for 1 hour 10 After ensuring that all of the acetone has evaporated add 50 pL of Protein Solubilization Solution to each tube Vortex thoroughly and incubate 10 minutes at room temperature 11 Add 450 uL of diluted Assay Diluent to each tube and vortex Note that samples have been diluted 1 10 from their original concentration 12 Centrifuge the tubes at 10 000 x g for 10 minutes to remove excess debris 5 A CELL BIOLABS INC 13 Determine the protein concentration of each sample 14 Prepare a standard curve as outlined in Table 1 15 Transfer 100 pL of each sample or prepared standards to a 96 well black fluorescence microtiter plate 16 Read the fluorescence with a fluorescence plate reader with 480 nm excitation 530 nm emission filter settings Calculation of Resul
7. protein concentrations and is only recommended if it can be prepared in the 1 10 mg mL range Use 1X Sample Diluent for all dilutions Preparation of the Fluorophore Standard Curve 1 Use the Fluorophore Standard to prepare a series of standards Prepare standards just prior to use Prepare a 25 uM solution of the Fluorophore Standard by diluting the stock 1 800 with 1X Assay Diluent Vortex thoroughly For example add 2 L of the 20 mM Fluorophore Standard stock to 1598 uL of 1X Assay Diluent 4 cet siotass ING 2 Prepare a 1 2 dilution series of Fluorophore standards in the concentration range of 0 nM 1 250 nM by diluting the 200 pM Fluorphore solution in 1X Assay Diluent see Table 1 Standard Fluorophore Assay Diluent Fluorophore Tubes uL uL nM 1 50 of 25 uM solution 950 1250 2 500 of Tube 1 500 625 k 500 of Tube 2 500 313 4 500 of Tube 3 500 156 5 500 of Tube 4 500 78 6 500 of Tube 5 500 39 T 500 of Tube 6 500 20 8 0 500 0 Table 1 Preparation of Fluorophore Standards Assay Protocol 1 Determine the protein concentration of each sample Adjust the protein concentration to between 1 10 mg mL with 1X Sample Diluent Add 50 uL of sample to a microcentrifuge tube and follow with a 50 pL of the freshly prepared 1X Protein Carbonyl Fluorophore solution to each sample tube Vortex thoroughly ie 3 Incubate samples overnight at room temperature and pr
8. r to homogeneity Store the 1X TCA at room temperature Note TCA Solution is highly corrosive Use caution when handling 1X Sample Diluent Dilute the 10X Sample Diluent to 1X with deionized water Stir to homogeneity Store the 1X Sample Diluent at 4 C 1X Assay Diluent Dilute the Assay Diluent 1 4 with deionized water Mix to homogeneity Store the 1X Assay Diluent at 4 C CELL BIOLABS INC e 1X Protein Carbonyl Fluorophore Prepare only enough for immediate applications Prepare 1X Protein Carbonyl Fluorophore by diluting the 100X Protein Carbonyl Fluorophore stock solution 1 100 with 1X Sample Diluent Do not store diluted solution Preparation of Samples All samples should be assayed immediately or stored at 80 C The assay can be used on cell lysates tissue homogenates serum plasma urine as well as other biological fluids when applicable Run proper controls as necessary Always run a standard curve with samples Use 1X Sample Diluent for dilutions and preparations of samples Sample concentrations must be adjusted to 1 10 mg mL prior to performing the assay Note For protein samples lower than 1 mg mL concentrate by centricon filtration or by mixing 0 8 vol of protein sample and 0 2 vol of 5X TCA solution incubating 10 minutes on ice and centrifuging at 10 000 g Dissolve the protein pellet in a small volume of IX PBS Optional High nucleic acid can erroneously contribute to higher estimation of car
9. s a convenient system to measure the protein carbonyl content in plasma serum cell lysates tissues and purified proteins Each kit provides sufficient reagents to perform up to 100 assays including unknown protein samples assay blanks and standards Assay Principle Protein carbonyls in protein samples 1 10 mg mL are first derivatized with the Protein Carbonyl Fluorophore The Fluorophore binds to the protein carbonyl group in a 1 1 ratio Proteins are then TCA precipitated and free Fluorophore is removed by washing the protein pellet with acetone After dissolving the protein pellet in GuHCl the absorbance of protein fluorophore product is measured fluorometrically with a 485 538 nm filter set and the protein carbonyl is subsequently calculated Related Products 1 STA 303 OxiSelect Nitrotyrosine Protein Immunoblot Kit 2 STA 304 Protein Tyrosine Nitration Control Nitrotyrosine BSA 3 STA 305 OxiSelect Nitrotyrosine ELISA Kit 4 STA 308 OxiSelect Protein Carbonyl Immunoblot Kit 5 STA 309 Oxidized Protein Immunoblot Control Carbonyl BSA 6 STA 310 OxiSelect Protein Carbonyl ELISA Kit 7 STA 315 OxiSelect Protein Carbonyl Spectrophotometric Kit 8 STA 318 OxiSelect AOPP Assay Kit 9 STA 816 OxiSelect N epsilon Carboxymethyl Lysine CML Competitive ELISA Kit 10 STA 817 OxiSelect Advanced Glycation End Products AGE Competitive ELISA Kit 2 cet sioLass INC Kit Components 1
10. the solubilized protein was determined by BCA assay For example for the 10 mg mL Oxidized BSA sample the solubilized protein concentration was 6 3 mg mL and its adjusted RFU reading is 1015 after 10 fold dilution with Assay Diluent Protein Carbonyl Concentration nM 1015 RFUs 4 9382 y intercept 1 3797 slope x 10 Dilution Factor 7321 nM Protein Carbonyl Content nmol mg 7321 nM 6 3 mg mL 7 321 nmol L 6 3 mg mL 1 162 nmol mg 8000 7000 6000 5000 4000 3000 2000 1000 0 ss E BSA 0x 10 mg mL m BSA r 10 mg mL E BSA ox 5 mg mL mBSA r 5mg mL mBSA ox 1 mg mL mBSA r 1 mg mL Protein Carbonyl nM Figure 2 Protein Carbonyl Contents in Oxidized and Reduced BSA _CELL BIOLABS INC References 1 Buss H Chan TP Sluis KB Domigan NM and Winterbourn CC 1985 Free Radic Biol Med 23 361 6 2 Cadenas E Boveris A Ragan CL and Stoppani AO 1977 Archives of Biochemistry amp Biophysics 180 248 257 3 Hammer M Schweitzer D Richter S Konigsdorffer E 2005 Physiol Meas 26 N9 N12 4 Reznick AZ and Packer L 1994 Methods Enzymol 233 263 357 5 Talent JM Kong Y and Gracy RW 1998 Anal Biochem 263 31 38 6 W akeyarga H Takeshige K Takayanagi R and Minakami S 1982 Biochem J 205 593 601 Recent Product Citations I Lauritzen K H et al 2015 Impaired dynamics and function of mitoch
11. ts The following figures demonstrate typical Protein Carbonyl Fluorometric Assay results Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer Molecular Devices with a 485 538 nm filter set and 530 nm cutoff One should use the data below for reference only This data should not be used to interpret actual results 2000 1800 1600 1400 1200 1000 800 600 400 200 0 y 1 3797x 4 9382 R 0 9996 RFUs o 200 400 600 800 1000 1200 1400 Fluorophore Standard Concentration nM Figure 1 Protein Carbonyl Fluorophore Standard Curve Calculations 1 Determine the average fluorescence for each standard and sample Subtract the value of 0 nM Fluorophore blank from itself and all other standard and sample values These are the adjusted RFU values 2 Graph the adjusted RFU values for the standards as a function of their corresponding concentration from Table 1 6 CELL BIOLABS INC 3 Determine the carbonyl concentration of samples using the equation obtained from the linear regression of the standard curve Calculation Example Chemically oxidized and reduced BSA were prepared at 10 5 and 1 mg mL in 1X Sample Diluent Samples were then derivatized with the Protein Carbonyl Fluorophore and processed according to the Assay Protocol After TCA precipitation and wash the protein pellet was dissolved in Protein Solubilization Solution and the protein concentration of
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