Apoptosis and Cell Proliferation (522915), страница 8
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Cross-reactions with other caspases are not known.Can be used to assay:P Lysates of adherent cells, of cells in suspension culture, of cells obtained exvivo or recombinant caspase 3.Kit contents1. Coating buffer, 10x2. Anti-caspase-3, 20x3. Blocking buffer, ready-to-use4. Incubation buffer, 5x5. DTT, 100x6. Substrate solution Ac-DEVD-AFC,20x7. AFC8. Positive control, apoptotic U937 celllysate9. MTP modules (12 x 8 wells)10.
Adhesive plate coverTypical results: see Figures 11–12Measure fluorometrically (excitation filter 400(370–425) nm and emission filter 505 (490–530) nm)Optional: for calibration, set up a calibration curve withdifferent dilutions of AFC as standardG Flow Chart 3: Assay procedure, Caspase 3 ActivityAssay.18The caspase 3 activity assay has been usedto detect caspase 3 activation in U937 cellsexposed to different concentrations of theapoptosis inducing agent camptothecin(Figure 11, dose response curve).
In thismodel system, the induction of apoptotis inonly 5% of U937 cells is sufficient for detection of caspase 3 activation. Caspase 3activity/fluorochrome development is proportional to the percentage of apoptoticcells.8045403082562015% annexin-V positive cellsAFC concentration [µM]35104102500.010.101.00concentration [µg/ml CAM]010.00G Fig.11: Dose-response experiment analyzed bythe caspase 3 Activity Assay. U937 cells were exposedto different concentrations of camptothecin for 4 h at37°C. Lysates were analyzed for caspase 3 activity andstandardized values are plotted versus concentration.Additionally, an aliqot of the same cells was analyzed forannexin-V binding.70Caspase 3Annexin-V6035040230% annexin-V positive cellsCaspase 3Annexin-V124Caspase 3 activity: fluorescence (Ex390 nm/Em505 nm)141201100001234Time [h]56Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)7G Fig.12: Kinetic study of caspase 3 activation bycamptothecin exposure in U937 cells.
U937 cells wereexposed to 4 µg/ml camptothecin for different timeintervals at 37°C. Lysates were analyzed for caspase 3activity and fluorescence (minus fluorescence of blank)is plotted versus time. Additionally, an aliqot of the samecells was analyzed for annexin-V binding in parallel.19Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)Anti-PARPCat.
No. 1 835 2381100 µl (50 blots)TypePolyclonal antiserum, from rabbitUseful forDetection on Western blots of PARP cleaved by caspases during early stagesof apoptosisSamplesCrude cell extractsMethodWestern blot of apoptotic cell extracts, followed by indirect immunodetectionof PARP cleavage fragmentTimeApprox. 5.5 h (immunodetection only)Significance of reagent: Anti-PARPrecognizes Poly-ADP-Ribose-Polymerase(PARP), a 113 kD protein that binds specifically at DNA strand breaks. PARP is alsoa substrate for certain caspases (for example, caspase 3 and 7) activated during earlystages of apoptosis. These proteases cleavePARP to fragments of approximately 89kD and 24 kD.
Detection of the 89 kDPARP fragment with Anti-PARP thusserves as an early marker of apoptosis.Test principle: The Anti-PARP antibodymay be used to detect the 89 kD PARPfragment (and intact PARP) from apoptotic cell extracts on a Western blot. The procedure (Flow Chart 4) involves:120Preparing crude extracts of apoptoticcells (for instance, by sonication and incubation of 105–107 cells in the presenceof urea, 2-mercaptoethanol, and SDS).2Separating proteins in the crude cell extracts on an SDS-polyacrylamide gel.3Transferring the separated proteins to amembrane by electroblotting.4Detecting PARP fragments (and intactPARP) on the membrane with the AntiPARP antibody.5Visualizing the antibody-protein complexes with an enzyme-conjugated antirabbit IgG secondary antibody and achromogenic or chemiluminescent enzyme substrate (see Table 3).Prepare crude extracts from apoptotic cells inextraction buffer (approx.
15 min, RT; then 15 min,65°C)Separate proteins in crude extracts by SDS-PAGEElectroblot proteins to nitrocellulose or PVDFmembrane (approx. 1 h, RT)Incubate membrane with blocking buffer (1 h, RT)Incubate membrane with Anti-PARP (diluted 1:2000)(2 h, RT)Wash membrane twice with blocking buffer(25 min, RT)Visualize antibody-antigen complexes with secondaryantibody and chromogenic/chemiluminescentdetection (approx. 2 h, RT)G Flow Chart 4: Assay of caspase activity withAnti-PARP.Antibody supplied as: Polyclonal antiserum from rabbit, stabilized.1Can be used to assay:P Crude cell extractsTypical results: See Figure 13.34113 kDSensitivity: PARP cleavage fragmentsfrom 3 x 105 apoptotic cells could be detected on a Western blot (Figure 13).Specificity: On Western blots, Anti-PARPrecognizes intact PARP from primates orrodents, as well as the large PARP fragmentgenerated by caspases.
Anti-PARP will immunoprecipitate intact PARP from primates or rodents.289 kDPARPG Figure 13: Detection of cleaved PARP in cell extracts of apoptotic CEM T cells. CEM T cells were incubated with one of three apoptosis-inducing drugs. Cellextracts from 3 x 105 treated or untreated cells were fractionated on an 10% polyacrylamide gel in the presence ofSDS.
After electrophoresis, proteins on the gel weretransferred to a PVDF membrane by electroblotting andthe blot was blocked with 5% powdered milk. Theblocked membrane was incubated with a 1:3000 dilutionof Anti-PARP. Subsequent incubations with a peroxidaseconjugated anti-rabbit secondary antibody and a peroxidase substrate revealed the presence of PARP cleavageproducts on the blot. Note that the antibody recognizesboth uncleaved PARP (113 kD) and the larger cleavagefragment (89 kD).Lane 1: Untreated control cellsLane 2: Cells treated with 100 ng/ml doxorubicin for 24 hLane 3: Cells treated with 1 mg/ml methotrexate for 24 hLane 4: Cells treated with 1 mg/ml cytarabin for 24 h.(Data is courtesy of Dr. Ingrid Herr, German CancerResearch Institute, department of molecular oncology,Heidelberg, Germany)ProductCat.
No.BM Chromogenic Western Blotting Kit (Mouse/Rabbit)1 647 644for 2000 cm2 membraneBM Chemiluminescence Western Blotting Kit(Mouse/Rabbit)1 520 709for 2000 cm2 membraneAnti-Rabbit IgG-peroxidase (POD), from sheep1 238 850200 unitsAnti-Rabbit IgG-alkaline phosphatase (AP), from sheep1 214 632500 units (1 ml)BM Chemiluminescence Blotting Substrate (POD)1 500 7081 500 694for 1000 cm2 membranefor 4000 cm2 membraneCSPD® (chemiluminescent AP substrate), ready-to-use1 755 6332 x 50 mlCDP Star™ (chemiluminescent AP substrate)ready-to-use1 685 6271 759 0511 ml2 x 1 mlBM Blue POD Substrate, precipitating1 442 066100 mlBM Teton POD Substrate, precipitating1 544 845200 mg (4 ml)BM Purple AP Substrate, precipitating1 442 074100 mlCell Death – Apoptosis and NecrosisApoptosis Assay MethodsAssays that measure apoptosis-induced proteases (caspases)1Pack SizeG Table 3: Related products for visualization of Anti-PARP.21Summary of methods for studying apoptosis in cell populationsSummary of methods for studying apoptosis in cell populations1.2.1.3 Summary of methods for studying apoptosis in cell populations.Method/Roche Molecular Biochemicals productParameteranalyzedLabelAssay principleAdvantagesLimitationsDNA Fragmentation Assay, radioactive11, 12DNA fragmentation(LMW and HMWDNA)[3H]-TdR or[125I]- UdR, prelabelP DNA fragments are released from the cytoplasm of apoptoticcells after lysis with non-ionic detergent.P The LMW DNA is separated from nuclear HMW DNA bycentrifugation.P The radioactivity in the supernatant and in the pellet isdetermined by LSC.P Quantitative measurement over a large range (several orders ofmagnitude)P Sensitive (103–104 cells/test required)P Suitable for analysis of cell-mediated (cytotoxicity) effectsPPPPDNA Fragmentation Assay,non-radioactive13DNA fragmentation(LMW DNA)BrdU, prelabelP DNA fragments are released from the cytoplasm of apoptoticcells after lysis with a non-ionic detergent.P The LMW DNA is separated from nuclear HMW DNA bycentrifugation.P The supernatant is analyzed by ELISA.PPPPPJAM Test14DNA fragmentation(HMW DNA)[3H]-TdR, prelabelP Cells are harvested by vacuum aspiration onto glass fiberfilters.
While LMW-DNA is washed through the filters, the HMWDNA is retained on these filters.P The radioactivity retained on the filters is measured by LSC.P Sensitive (103–104 cells/test required)P Only 1 washing step required for the labeled cellsP Low spontaneous release: cytotoxic events causing low cell lysis overprolonged period of time (8–24 h) can be studiedP Optimal for microtiter plate formatPPPPRadioactive isotopePrelabeling of the target cells requiredLimited to target cells proliferating in vitroIn apoptotic cells, DNA is only partially lost: viable and damaged cellsare separated by only a narrow range of assay valuesAlkaline Elution Analysis15DNA fragmentation(LMW and HMWDNA)[3H]-TdR, prelabelP Cells are loaded onto polycarbonate filters.P The filters are incubated with three different buffer solutionscontaining SDS, pH 10, SDS + Proteinase K, pH 7, or SDS,pH 12.3.P The radioactivity in each fraction (LMW DNA) as well as theradioactivity retained on the filter (HMW DNA) is quantifiedby LSC.P Differential elution allows the detection of strand breaks in DNA,DNA-interstrand crosslinks and DNA-protein crosslinksPPPPPRadioactive isotopePrelabeling and washing of the target cells requiredLimited to target cells proliferating in vitroInsensitive (106 cells/test required)Labor-intensive and time-consuming:only a few tests may be performed simultaneouslyDNA Ladder Assay16(LMW and HMW DNA by size)DNA fragmentationnoneP Cellular DNA is isolated by extraction and quickly purified.P Purified total DNA (LMW and HMW DNA) is analyzed by agarosegel electrophoresis and visualized by staining with ethidiumbromide.P Hallmark of apoptosis: demonstration of the mono- and oligonucleosomal DNA fragments (180 bp multimers)P No prelabeling of the cells required: not limited to cells whichproliferate in vitroP Non-radioactiveP No quantitative measurementP Insensitive: More than 106 cells/test requiredP Labor-intensive and time-consuming:only a few tests may be performed simultaneouslyDNA fragmentation(LMW DNA inassociation withhistones)noneP Histone complexed DNA-fragments (mono- and oligonucleosomes, LMW DNA) are released from the cytoplasm ofapoptotic cells after lysis.P The LMW DNA is separated from nuclear HMW DNA bycentrifugation.P The supernatant is analyzed by ELISA.P Sensitive (102–104 cells/test required)P No prelabeling of the cells required: not restricted to cells whichproliferate in vitroP Non-radioactiveP Detection of DNA and histones in one immunoassay demonstratesmono- and oligonucleosomal DNA fragmentsP Samples have to be analyzed immediately because storage reduces page 13ELISA signalof this guideP Not recommended for tissue homogenates.
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