PROTOCOL Cmplex II Enzyme Activity Micrplate Assay Kit 1850 Millrace Drive, Suite 3A Eugene, Oregn 97403 MS241 Rev.0 DESCRIPTION Cmplex II Enzyme Activity Micrplate Assay Kit Sufficient materials are prvided fr ne 96-well micrplate. Kit Cntents: Item MS241 20X Buffer 15 ml 10X Detergent 2 x 1 ml 10X Blcking Slutin 5 ml Cmplex II Activity Buffer 25 ml DCPIP 250 µl Succinate 500 µl Ubiquinne 2 60 µl Lipid Mix 6 ml Pre-cated 96-well micrplate (12 strips) 1 INTRODUCTION Cmplex II, als knwn as succinate-cenzyme Q reductase (SDH, EC 1.3.5.1), is ne f the five cmplexes invlved in xidative phsphrylatin in the inner mitchndrial membrane and als a member f the tricarbxylic acid cycle (TCA). It catalyzes electrn transfer frm succinate t the electrn carrier, ubiquinne, but unlike the ther fur cmplexes it is nt a prtn pump. The prduct ubiquinl is utilized by cmplex III in the respiratry chain and the prduct fumarate is necessary t maintain the TCA cycle. Succinate + ubiquinne (Q) Fumarate + ubiquinl (QH 2 ) The Cmplex II Enzyme Activity Micrplate Assay (MS241) is designed fr determining the Cmplex II activity in a sample. Each f the 96 wells in the kit has been cated with an anti-cmplex II mnclnal antibdy (mab) which purifies the enzyme frm a cmplex sample such as mitchndria, tissue hmgenate r cell lysate. After this in-well purificatin the prductin f ubiquinl by the enzyme is cupled t the reductin f the dye DCPIP (2,6-diclrphenlindphenl) and a decreases in its absrbance at 600 nm which in turn recycles the substrate ubiquinne, as shwn belw. ubiquinl (QH 2 ) + DCPIP ubiquinne (Q) + DCPIPH 2 blue clrless
Nte: This prtcl cntains detailed steps fr measuring Cmplex II activity. Be cmpletely familiar with the prtcl and the Ntes sectin (page 9) befre beginning the assay. D nt deviate frm the specified prtcl steps r ptimal results may nt be btained. The prtcl has 3 steps: A. Sample preparatin B. Plate lading C. Measurement This micrplate assay (Catalg # MS241) has been develped fr use with human samples. Hwever, the kit als wrks with bvine, muse and rat samples. Other species are untested at this time. This assay is designed fr use with hmgenates frm cultured cells, and islated mitchndria - 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 (50 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. Example Ranges: Bvine heart mitchndria 1-25 µg/well 20-500 µg/ml Whle cultured cell extract 4-250 µg/well 80-5000 µg/ml NOTE: Ranges fr tissue extract may vary slightly. The lwest amunt indicated is the lwest amunt tested (>2x backgrund). Fr sample lading use the recmmended amunt specified n page 3. Intra assay variatin = <15 % ADDITIONAL MATERIALS REQUIRED Spectrphtmeter plate reader (Mlecular Dynamics SpectraMax recmmended) capable f measuring absrbance at 600 nm. Methd fr determining prtein cncentratin Deinized water Multichannel pipette PBS (phsphate buffered saline) fr recipe see /PDF/western.pdf Page 2 f 9
COMPLEX II ACTIVITY ASSAY Nte: This prtcl cntains detailed steps fr measuring Cmplex II activity. Be cmpletely familiar with the prtcl befre beginning the assay. D nt deviate frm the specified prtcl steps r ptimal results may nt be btained. When ding multiple experiments it is recmmended t make nly prprtinally enugh wrking slutins and reagents frm the supplied cncentrated stcks. A. Sample preparatin 1. Prepare the buffer slutin by adding 15 ml f 20X buffer t 285 ml deinized H 2 O. Label this 1X Buffer slutin. Mix, stre in the refrigeratr. 2. Prepare the Incubatin slutin by adding 5 ml 10X Blcking Slutin t 45 ml 1X Buffer. Label this 1X Incubatin slutin. Mix, stre in the refrigeratr. 3. Resuspend sample tissue hmgenate, cell culture pellet, r mitchndria t 5.5 mg/ml in PBS. 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). Therefre the final prtein cncentratin is 5 mg/ml. 5. Mix immediately and incubate fr 30 minutes n ice. 6. Centrifuge at 25,000 g fr 20 minutes at 4 C. Cllect the supernatant discard the pellet. B. Plate Lading 7. Dilute the sample in 1X Incubatin slutin, t the desired cncentratin per ml shwn in the table belw. Sample Type Recmmended amunt Whle cultured cell extract 60 µg/50 µl (1.2 mg/ml) Heart mitchndria 10 µg/50 µl (0.2 mg/ml) 8. Keep the diluted samples n ice until ready t begin. 9. Add 50 L f diluted sample per well. Be sure t include a nrmal r cntrl sample and als a buffer nly sample (1X Incubatin slutin). A dilutin series f a nrmal cntrl sample is als recmmended. 10. Cver the plate and incubate fr 2 hurs at rm temperature. C. Measurement 11. The bund mnclnal antibdy has immbilized the enzyme in the wells. Empty the wells by turning the plate ver and shaking ut any remaining liquid. 12. Once emptied, add 300 μl f 1X Buffer slutin t each well used. Page 3 f 9
13. Empty the wells again and add anther 300 μl f 1X Buffer slutin t each well used. 14. Empty the wells and add 40 µl f lipid slutin t each well used. Incubate 30 minutes. 15. Prepare the Activity slutin. Make nly enugh slutin prprtinal t the number f micrplate strips used. Use the fllwing table t calculate hw much slutin t make fr 1-12 micrplate strips used. N. f micrplate strips Ubiquinne 2 (µl) Succinate (µl) DCPIP (µl) 1 5 42 21 2.1 2 10 83 42 4.2 3 15 125 63 6.3 4 20 167 83 8.3 5 25 208 104 10.4 6 30 250 125 12.5 7 35 292 146 14.6 8 40 333 167 16.7 9 45 375 188 18.8 10 50 417 208 20.8 11 55 458 229 22.9 12 60 500 250 25 Activity buffer (ml) 16. D nt empty the wells; instead add 200 µl f Activity slutin (A15) t each well already cntaining 40 µl lipids fr a ttal f 240 µl. Any bubbles in the wells shuld be ppped with a fine needle as rapidly as pssible, begin micrplate reading using the fllwing parameters: Activity - Kinetic measurement OD 600 nm Rm temperature Time: 60 mins Interval: 20 sec - 1 min NO shake befre r between readings Save data and analyze as described in the DATA ANALYSIS sectin. Nte it is pssible t make an endpint measurement in place f kinetic measurement, but be sure t measure the endpint befre the mst active sample has begun t slw dwn (see example belw BHM sample >1600s). Page 4 f 9
DATA ANALYSIS COMPLEX II ACTIVITY The initial slutin fr the activity measurement shuld be blue in appearance with an OD f apprximately 0.2 mod units at 600 nm. The reductin f ubiquinne and subsequent reductin f DCPIP is measured as a decrease in absrbance at OD 600 nm (see Figure 1). Mnitr the rate f decrease in absrbance at 600 nm ver time. Calculate the rate between tw time pints fr all the samples where the decrease in absrbance is the mst linear (typically between 15 mins and 25 mins shwn belw). After 30 minutes the rate f reductin in absrbance may decline fr the mst active samples due lack f substrate s d nt calculate the rate after this pint. Rate (mod/min) = Absrbance 1 - Absrbance 2 Time (min) The activity f immuncaptured Cmplex II is the mean f measurements btained with immuncaptured enzyme minus the rate btained withut immuncaptured enzyme. Fr example, if the rates f immuncaptured Cmplex II are 3.2, 3.1 and 3.7 mod/min and the backgrund rate (null sample) is 0.1 mod/min, the activity f Cmplex II is (3.2+3.1+3.7)/3-0.1 which is 3.23 mod/min. Nw the activity f immuncaptured Cmplex II in between samples can be cmpared. Figure 1. Example f raw data. Nte the lag perid befre activity. Als nte the activity f mitchndria (BHM, bvine heart mitchndria) is higher than whle cell lysate (HepG2, human hepatblastma) and the reactin ends at >1600 secnds because the substrates are used up. This assay is cmpatible with different sample types such as mitchndria, tissue r cell lysates and in multiple species including human and rdent samples. Typical linear range data are shwn belw in Figure 2. Page 5 f 9
Figure 2. Data are mst easily interpred by wrking in the linear range f the assay as shwn here, hwever the range can als be extended by nn-linear curve fitting. Page 6 f 9
FLOW CHART Fr quick reference nly. Be cmpletely familiar with previus details f this dcument befre perfrming the assay. Prepare Sample (1-2 hurs) Bring sample t 5.5 mg/ml in PBS. Perfrm detergent extractin with 1/10 vlume detergent, 30 mins n ice, fllwed by 25,000 g centrifugatin fr 20 minutes at 4 C. Take supernatant. Adjust cncentratin t recmmended dilutin fr plate lading in Incubatin buffer. Lad Plate (2 hurs) Lad sample(s) n plate being sure t include psitive cntrl sample and buffer cntrl as a null reference. Incubate 2 hurs at rm temperature. Measure (1 hurs) Rinse wells twice with 1X Buffer. Add 40 μl f Lipid mix t wells Add 200 µl Activity slutin int the lipid mix in each well. Measure OD 600 at 1 minute intervals fr 1 hur at rm temperature with n plate shake functin. 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 MS241 Page 8 f 9
NOTES Sample preparatin is crucial t a successful analysis. Key parameters: Hmgenizatin Samples must be cmpletely hmgenus. Fr cultured cells this shuld nly require pipetting up and dwn t break apart clumps f cells. Similarly fr mitchndrial preparatins, pipetting is enugh t distribute the mitchndria evenly in slutin. Fr sft tissue, and especially fr hard tissues such as muscle, thrugh hmgenizatin must ccur. This is best accmplished with a hand held tissue grinder such as an electric Ultra Turrax T8 tissue grinder r a Dunce glass tissue grinder available frm MitSciences. It is recmmended t use ne f MitSciences Mitchndrial Islatin Kits (MS850- MS853). Sample slubilizatin It is mst cnvenient t resuspend t apprximately 10 mg/ml. Then determine the exact prtein cncentratin by BCA methd (Pierce). Then add slutin t a prtein cncentratin f 5.5 mg/ml in PBS. The sample can nw be extracted by adding 1/10 vlume f the supplied detergent. The final prtein cncentratin is nw 5 mg/ml, which is the ptimal cncentratin fr intact Cmplex II slubilizatin by the supplied detergent. The sample is incubated, centrifuged and supernatant (detergent extract) is cllected. Inhibitr sensitivity The enzyme is sensitive t 2-thenyltrifluracetne (TTFA) a specific inhibitr f Cmplex II with IC 50 f 30 µm. Page 9 f 9