PROTOCOL SOD2 Prtein Quantity Micrplate Assay Kit 1850 Millrace Drive, Suite 3A Eugene, Oregn 97403 MS746 Rev.0 DESCRIPTION SOD2 Prtein Quantity Micrplate Assay Kit Sufficient materials are prvided fr 96 measurements (ne 96-well micrplate). Kit Cntents: Item MS746 20X Buffer (Tube 1) 20 ml Detergent 1 ml 20X Detectr Antibdy (Tube A) 1 ml 20X HRP Label (Tube B) 1 ml 10X Blcking Buffer (Tube 2) 8 ml Develpment Slutin (Tube 3) 20 ml 96-well micrplate (12 strips) 1 The 96-well micrplate has a mnclnal antibdy pre-bund t the wells. This plate can be separated int twelve 8-well strips and thus can be used fr up t 12 individual experiments. Strage: Stre all cmpnents at 4 C. The kit is stable fr at least 6 mnths. INTRODUCTION Aerbic rganisms have several mechanisms t prtect against xidatin, particularly frm harmful reactive xygen species (ROS). Superxide and its prducts have been implicated in a wide range f diseases including cancer, inflammatin, neurdegenerative diseases, diabetes and aging itself. The principle cellular anti-xidants are the superxide dismutase family (SOD, E.C. 1.15.1.1). These enzymes dismutate superxide int hydrgen perxide which is further detxified by ther cellular defenses such as glutathine perxidase and catalase. The SOD family has 3 members, tw f which are Cu-Zn type: the extracellular SOD3 and the cytplasmic SOD1. The ther member is the mitchndrial Mn type SOD2. The mitchndrial Mn-SOD2 is a hmtetramer f subunit mass 23 kda in the mitchndrial matrix with ne manganese in per subunit. SOD2 is vital since SOD1 knckut mice appear t develp nrmally while SOD2 knckut mice d nt and die shrtly after birth. SOD2 can als be the target f xidative stress Tyr34 near the Mn active site can becme nitrated leading t inactivatin. It has been pstulated that an increased reactive nitrgen species in ALS may cntribute t site-specific SOD2 inactivatin and exacerbatin f ALS pathgenesis. SOD2 levels may be dwn-regulated in tumr cells and recent bservatins shw that ver expressin f SOD2 in tumr cells may suppress cell divisin and cancer grwth (Oberley, Bimedecine & Pharmactherapy, 2005, 59, p143-8).
Nte: This prtcl cntains detailed steps fr measuring SOD2 quantity. Be cmpletely familiar with the prtcl and the FAQ sectin (page 8) befre beginning the assay. D nt deviate frm the specified prtcl steps r ptimal results may nt be btained. The prtcl has 4 steps: A. Sample preparatin B. Plate lading C. Add detectin antibdies D. Quantity measurement This micrplate assay (Catalg # MS746) has been develped fr use with human samples. Muse and rat samples may be used in the prvided sample range guidelines. This assay is designed fr use with hmgenates frm cultured cells. Islated mitchndria and tissue lysates can als be used, but sme sample ptimizatin may be necessary. As described belw, hmgenized samples shuld be resuspended t 5.5 mg/ml prtein. The prteins are detergent extracted and laded t within the linear range f the assay (see belw). A cntrl r nrmal sample shuld always be included in the assay as a reference. Als, include a null r buffer cntrl t act as a backgrund reference measurement. Typical linear ranges per well (200 µl) and per milliliter are listed belw. The ranges may be extended by using a nn-linear fit f the data frm a nrmal sample. Typical Linear Ranges: Human heart mitchndria 1-10 µg/well 5-50 µg/ml Human liver mitchndria 1-10 µg/well 5-50 µg/well Whle cultured HepG2 extract 2-25 µg/well 10-125 µg/ml Whle cultured fibrblast cell extract 12-100 µg/well 5-500 µg/ml Muse brain mitchndria 25-100 µg/well 125-500 µg/well NOTE: SOD2 levels between cell types als vary greatly. Ranges fr mitchndria are dependent upn mitchndrial purity and integrity. Fr sample lading use the recmmended amunt specified n page 3. Intra assay variatin = 12% (n=48), inter assay variatin = 10.4 % (n=48) ADDITIONAL MATERIALS REQUIRED Spectrphtmeter plate reader (Mlecular Dynamics SpectraMax recmmended) capable f measuring absrbance at 600 nm (r 450 nm after additin f 1N HCl (nt supplied)). Methd fr determining prtein cncentratin Deinized water Multichannel pipette PBS (phsphate buffered saline fr recipe see /PDF/western.pdf Page 2 f 9
MICROPLATE ASSAY PROTOCOL A. Buffer and Sample Preparatin 1. Prepare the buffer slutin by adding 20 ml f 20X Buffer (Tube 1) t 380 ml deinized H 2 O. Label this Slutin 1. 2. Fr entire plate (12 strips) add 8 ml f 10X Blcking Buffer (Tube 2) t 72 ml f Slutin 1. Label this Incubatin Slutin. NOTE: When using fewer strips make prprtinately less (i.e. 0.67 ml Tube 2 + 6 ml Slutin 1 fr each strip used). 3. Determine the sample prtein cncentratin by a standard methd and then adjust the prtein cncentratin t 5.5 mg/ml in PBS. NOTE: If the sample is less than 5.5 mg/ml, centrifuge t pellet again and take up in a smaller vlume t cncentrate the pellet and repeat prtein cncentratin measurement. The ptimal prtein cncentratin fr detergent extractin is 5.5 mg/ml. 4. Add 1/10 vlume f Detergent t the sample (e.g. if the ttal sample vlume is 500 µl, add 50 µl f Detergent). The final prtein cncentratin is nw 5 mg/ml. 5. Mix immediately and then incubate n ice fr 30 minutes. 6. Spin in tabletp micrfuge at maximum speed (~20,000 g, ~16,000 rpm) fr 20 minutes. Carefully cllect the supernatant and save as sample. Discard the pellet. 7. The micrplate wells are designed fr 200 µl sample vlume per well, s dilute samples t the fllwing recmmended cncentratins by adding Incubatin Slutin : Sample Type Recmmended amunt Human whle cultured cell extract 20 µg/200 µl (100 µg/ml) Human Tissue / mitchndria extract 5 µg/200 µl (25 µg/ml) Nte: D nt use mre than 40 ml f the Incubatin Slutin fr sample dilutin. 8. Keep diluted samples n ice until ready t prceed. B. Plate Lading A. Add 200 µl f each diluted sample int individual wells n the plate. Include a cntrl (nrmal) sample as a psitive cntrl. Als include a buffer cntrl (200 µl Incubatin Slutin) as a null r backgrund reference. B. Incubate fr 3 hurs at rm temperature. Page 3 f 9
C. Add Detectin Antibdies 1. The bund mnclnal antibdy has immbilized the prtein in the wells. Empty the wells by turning the plate ver and shaking ut any remaining liquid. 2. Once emptied, add 300 µl f Slutin 1 t each well used. 3. Empty the wells again and add anther 300 µl f Slutin 1 t each well used. 4. Separately, add 1 ml 20X Detectr Antibdy (Tube A) t 20 ml f Incubatin Slutin. Label this Slutin A. When using fewer strips make prprtinately less Slutin A. 5. Empty the wells again by turning the plate ver and shaking ut the remaining liquid. 6. Add 200 µl f Slutin A t each well used. 7. Incubate at rm temperature fr 1 hur. 8. Separately, add 1 ml 20X HRP Label (Tube B) t 20 ml f Incubatin Slutin. Label this Slutin B. When using fewer strips make prprtinately less Slutin B. 9. Empty the wells again quickly by turning the plate ver and shaking ut the remaining liquid. 10. Add 300 µl f Slutin 1 t each well used. Empty the wells again and add anther 300 µl f Slutin 1 t each well used. 11. Empty the wells and add 200 µl f Slutin B t each well used. 12. Incubate at rm temperature fr 1 hur. D. Quantity Measurement 1. After 1 hur empty the wells by turning the plate ver and shaking ut any remaining liquid. 2. Add 300 µl f Slutin 1 t each well used. Repeat this step three mre times fr a ttal f 4 rinse steps. 3. Add 200 µl f Develpment Slutin t each well used. Any bubbles in the wells shuld be ppped with a fine needle as rapidly as pssible. 4. Place the plate in the reader and recrd with the fllwing kinetic prgram (alternatively an endpint measurement can be made by stpping the reactin at a user defined time by additin f 100 µl f 1N HCl per well. Kinetic measurement OD 600 nm Time: 30 mins Interval: 20 sec - 1 min Autshake between readings Endpint Measurement Add 100 µl 1M HCl/well Autshake befre reading Measure OD 450 nm 5. Save data and analyze as described in the DATA ANALYSIS sectin. Page 4 f 9
DATA ANALYSIS The quantity f SOD2 captured in each well is prprtinal t the amunt f HRP activity within each well. Therefre the relative quantity is the change in absrbance at 600 nm/minute/amunt f sample laded int the well. Examine the linear rate f increase in absrbance at 600 nm with time. This is shwn belw where the rate r slpe is calculated between tw time pints. Mst micrplate sftware is capable f perfrming this functin. Repeat this fr all samples. Fr the cntrl r nrmal sample, the rate versus amunt laded is pltted as a straight line in the linear regin f the assay as shwn abve. Cmpare the rates f the cntrl (nrmal) sample and with the rate f the null (backgrund) and with yur unknwns, experimental r treated samples t get the relative amunt f SOD2. Page 5 f 9
SOD2 levels were quantified in the whle cell extract frm three different cell lines; HepG2, HeLa and HDFN cells all grwn in glucse as well as HepG2 cells grwn in galactse. A striking difference is seen in expressin levels, this difference is cnfirmed in Western bltting. This difference may be due t increased mitchndrial respiratry chain activity in HepG2 galactse grwn cells and a resulting need fr mre antixidant enzyme activities. Example: 25 µg whle cell lysate analyzed by MS746 quantity SOD2 micrplate. Example Cnfirmatin by Western bltting. 15 µg whle cell lysate analyzed by an anti-sod2 Western blt. Page 6 f 9
FLOW CHART Fr quick reference nly. Be cmpletely familiar with the previus details f this prtcl befre perfrming the assay. Prepare Sample (1-3 hurs) Prepare 1X Slutin 1 and the 1X Incubatin Slutin. Dilute the sample t 5.5 mg/ml in PBS. Perfrm detergent extractin with 1/10 vlume detergent fllwed by 20,000 x g centrifugatin fr 20 min. Take supernatant. Dilute sample t desired cncentratin in Incubatin Slutin. Lad Plate (3 hurs) Lad sample(s) within specified range int wells. Be sure t include a nrmal as a psitive cntrl sample and buffer cntrl as null reference. Incubate 3 hurs at rm temperature. Antibdy Incubatin #1 (1 hur) Empty wells and wash each well twice with 300 µl Slutin 1. Add 200 µl f Slutin A t every well used. Incubate fr 1 hur at rm temperature. Antibdy Incubatin #2 (1 hur) Empty wells and wash each well twice with 300 µl Slutin 1. Add 200 µl f Slutin B t every well used. Incubate fr 1 hur at rm temperature. Measure (0.5 hurs) Empty wells, wash wells used fur times with 300 µl Slutin 1. Add 200 µl f Develpment slutin. Measure OD 600 at 1 minute intervals fr 30 minutes at rm temperature. Page 7 f 9
MICROPLATE MS / / A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 MS746 Page 8 f 9
Frequently Asked Questins 1. What is the minimum amunt f cells r tissue needed t accurately measure SOD2 quantity? SOD2 quantity varies frm cell line t cell line. A signal f 2x ver backgrund was seen with 2 µg and 6 µg fr Hepg2 and fibrblast cells respectively. This crrespnds t apprximately 20,000-100,000 cells per well. 2. Is it pssible t speed up this assay? Antigen-antibdy reactins are dependent n many cnditins such as temperature and mvement f mlecules. The higher the temperature and the faster the mvement f mlecules and the sner the saturatin f binding sites ccur. This assay can be perfrmed in abut half the time if sample, detectr antibdy and HRP label incubatin steps are carried ut at 37 C n a rtating platfrm. Hwever, it is crucial t be cnsistent with all assays fr crss-cmparisns. Under these specified cnditins, samples can be incubated fr 1.5 hurs and detectr and label incubatin times can be reduced t 35 minutes each. 3. Can I use this plate t determine SOD2 quantity in tissues frm rdents r ther animal mdels? Yes rat and muse tissues have been tested but may require mre material than is indicated fr human samples. 4. Which immungen was used t develp the antibdies used in this kit? Purified SOD2. 5. What evidence d yu have that the captured prtein is in fact pure SOD2? Immunprecipitatins using these antibdies were perfrmed n a large scale and analyzed by SDS-PAGE purity and Mass Spectrmetry fr identity. 6. Which is the exact epitpe that binds t the capture antibdy (attached t the plate) and the detectr antibdy prvided with this kit? These antibdies bind the native cnfrmatin f SOD2, the exact epitpes are unknwn. 7. Hw d I analyze tissue samples? Tissue samples must be hmgenus befre detergent extractin. Hmgenize tissue samples thrughly by using a Dunce hmgenizer (see MitSciences prduct MS851) r micrtissue grinder/ultraturrax T8. Measure prtein cncentratin by BCA prtein assay (ThermFisher- Pierce). Dilute t 5.5 mg/ml in PBS, then add 1/10 vlume f detergent fr extractin and prceed frm page 3 step A5. Page 9 f 9