Apoptosis and Cell Proliferation (522915), страница 14
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No. 1 922 4591100 testsTypeMonoclonal antibody, clone 2R2, IgG3, mouseUseful forDetection of the apoptosis-inducing Fas receptor by immunohistochemicalstaining, flow cytometry or Western blottingSamplesCell suspensions, adherent cells, paraffin-embedded tissue sections, crude cellextracts (Western blots)MethodPreparation of sample followed by indirect immunodetection of Fas moleculeTimeApprox. 1.5 h for flow (immunodetection only)Approx 4.5–5.0 h for immunohistochemical staining (dewaxing throughdetection) or immunodetection in Western blotsSignificance of reagent: The antibodymay be used for immunohistochemistryand FACS analysis of the Fas/Apo-1 molecule.
The Fas (CD95/Apo-1) molecule hasbeen identified as a cell surface receptorthat could induce apoptotic cell death oftransformed cells.Test principle: The antibody may be usedto detect the Fas/Apo-1 molecule by Western blot (Flow Chart 9), FACS analysis(Flow Chart 10) or immunohistochemicalstaining (Flow Chart 11). The processes involve:Western blot42FACS1Wash cells in PBS2Incubation with Anti-Fas-Biotin3Incubation with Streptavidin-Fluorescein (Cat.
No. 1 055 097)4Dilute cells in PBS for FACSImmunohistochemistry1Drying fixed cells2Dewaxing sample in xylol3Blocking of endogenous POD4Detection of Fas molecule with AntiFas-Biotin1Running sample on gel and transfer tomembrane2Incubation with Anti-Fas-Biotin, wash3Incubation with Streptavidin conjugate,wash5Incubation with Streptavidin-POD(Cat. No. 1 089 153).4Detection of Fas receptor using suitablesubstrate6Visualizing complex with DAB substrate (Cat. No.
1 718 096).Prepare samples and separate by SDS-PAGEBlot the proteins onto a solid matrix (e.g. PVDF) using astandard protocolBlock non-specific binding sites on membrane byincubating in blocking solution (e.g. 0.1 M Tris-HCl,0.1% BSA w/v, 0.1% Tween® 20 v/v)(1 h, RT, with gentle shaking)Incubate membrane with Anti-Fas-Biotin (concentration 1 µg/ml) in PBS containing 1% BSA(1 h, RT, with gentle shaking)Place fixed sections onto APES-coated slide and dry(overnight, 37°C)Dewax in xylol (2 x 10 min, RT) and rehydrate throughgraded alcoholMicrowave slides in citric acid buffer, 5 x 5 min athighest intensity, then cool to RTRinse slides in PBS and block in PBSA (PBS + BSA)(20 min, RT)1Perform endogenous POD blocking if necessary(30 min in methanol/0.3% H2O2 at RT)Wash membrane 2 x 10 min in TBST at RTIncubate membrane with streptavidin-conjugate asrecommended (we recommend Streptavidin-POD)Wash membrane 4 x 15 min in large volumes of TBSTIncubate membrane with DAB until desired colordevelopment (~5–15 min, RT)G Flow Chart 9: Western blot procedure withAnti-Fas-Biotin.Wash cells (monolayer or cell suspension) with PBSIncubate with Anti-Fas-Biotin (diluted 1:20)(45 min, 37°C)Add Anti-Fas-Biotin solution (diluted 1:20 to 1:100)(30 min, 37°C)Rinse slides in PBSA and incubate with StreptavidinPOD (10 µg/ml) (30 min, RT)Wash slides three times in PBSIncubate the slides with DAB substrate until clearvisible color developsCounterstain with hemalum, mount and viewG Flow Chart 11: Staining procedure, immunohistochemistry with Anti-Fas-Biotin.Antibody supplied as: Mouse monoclonalantibody (clone 2R2, IgG3), lyophilizate.Wash cells twice with PBSIncubate with Streptavidin-Fluorescein solution(10 µg/ml) 30 min, RTWash cells twice with PBSSpecificity: The antibody recognizes thehuman Fas/Apo-1 (CD95).Can be used to assay:P Adherent cells (monolayer on slides)P Cell suspensionsExamine underfluorescence microscopeDilute cells in PBS forFACS analysisCell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsP Paraffin-embedded sectionsP Crude cell extractsG Flow Chart 10: Assay procedure, flow cytometryand immunofluorescence with Anti-Fas-Biotin.43Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsAnti-Bcl-2 oncoprotein, humanCat.
No. 1 624 98911 mlTypeMonoclonal antibody, from mouseUseful forDetection of the apoptosis-suppressing Bcl-2 protein on slides or WesternblotsSamplesCytospins, cell smears, frozen or fixed paraffin-embedded tissue sections;crude cell extractsMethodPreparation of sample, followed by indirect immunodetection of Bcl-2 proteinTimeApprox. 3.5 h (immunodetection only)Significance of assay: The antibody maybe used to detect Bcl-2 in tissue or cells (byimmunohistochemistry or immunocytochemistry) or in crude cell extracts (onWestern blots). The product of the Bcl-2proto-oncogene is thought to be a negativeregulator (suppressor) of apoptosis inmammalian cells.
The 26 kD Bcl-2 proteinis found principally in lymphoid tissue andcells (e.g., lymph node, spleen, thymus, peripheral blood lymphocytes).Test principle: The antibody may be usedto detect Bcl-2 in situ by immunohistochemistry or immunocytochemistry. Thestaining process (Flow Chart 12) involves:Incubate the slide with Anti-Bcl-2 oncoprotein(diluted 1:40–1:80) (1 h, RT)Wash slide three times with PBSIncubate the slide with an Anti-mouse (bridging)antibody (1 h, RT)Wash slide three times with PBSIncubate the slide with APAAP solution (30 min, RT)Fixing the sample (frozen sections, freshcells) on a slide.Wash slide three times with PBS2Detecting Bcl-2 protein in the samplewith Anti-Bcl-2 antibody solution.Incubate the slide with Fast Red substrate until a strongcolor develops3Detecting the immobilized Anti-Bcl-2antibody with an anti-mouse (bridging)antibody.14544Fix sample (frozen tissue sections, fresh cell smears)on slide with acetone or methanol (10 min, –20°C)Binding the bridging antibody with alkaline-phosphatase-anti-alkaline phosphatase (APAAP) solution.Visualizing the antibody-antigen complexes with chromogenic alkaline phosphatase substrate (Fast Red tablets, Cat.No.
1 496 549).Counterstain with hemalum, mount and viewG Flow Chart 12: Assay procedure, immunohistochemistry/immunocytochemistry with Anti-Bcl-2.Antibody supplied as: Mouse monoclonalantibody (clone 124) in cell culture supernatant.Specificity: On Western blots, Anti-Bcl-2binds the 26 kD product of the Bcl-2 proto-oncogene.
In tissue, the antibody reactsstrongly with amino acids 41–54 of theBcl-2 protein on cytospins, cell smears, andfrozen lymphoid tissue sections. In routinely fixed, paraffin-embedded tissue, theantibody reacts with a lower percentageof cells, since the epitope is not alwayspreserved under these conditions.Can be used to assay:P CytospinsP Cell smearsP Frozen tissue, some paraffin-embeddedtissueP Crude cell extracts (on Western blots)Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteins145Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsAnti-p53-Protein, mutantMonoclonal antibody100 µgCat.
No. 1 696 823Anti-p53-Protein panMonoclonal antibody100 µgCat. No. 1 413 1471Anti-p53 panPolyclonal antibody200 µgCat. No. 1 810 928Anti-p53, Biotin labeledPolyclonal antibody150 µgCat. No. 1 810 936Anti-p53, Peroxidase labeledPolyclonal antibodyCat. No. 1 810 94450 USignificance of reagents: The antibodies are suitable for the detection of the p53 proteinin immunohistochemistry and Western blot.AntibodyRecognizes p53fromCloneIg ClassTypical resultsAnti-p53-Protein,mutanthuman, mouse, rat,hamster, monkey,cow, chickenPAb240mouse IgG1Anti-p53-Proteinpanhuman, mouse, rat,hamster, monkeyPAb122mouse IgG2bSee Figure 29*Anti-p53 panhuman, mouse, rat,hamster, monkey,cow, chickenpolyclonal serumsheep IgGSee Figure 30Anti-p53, Biotinlabeledhuman, mouse, rat,hamster, monkey,cow, chickenpolyclonal serumsheep IgGAnti-p53, Peroxidase labeledhuman, mouse, rat,hamster, monkey,cow, chickenpolyclonal serumsheep IgGG Table 12: Anitbodies to p53*For another example of how this antibody can be used in the laboratory, see Del Bufalo, D.
et al. (1996) J. Clin. Invest.98, 1165–1173.46G Figure 29: Detection of p53 in a human ovarianadenocarcinoma with a monoclonal antibody.A cryostat section was prepared from a partially differentiated, papillary, advanced ovarian carcinoma. The section was stained with 5 fg/ml of mouse monoclonal antip53 pan (Cat. No. 1 413 147). A biotinylated rabbit antimouse antibody was used as secondary antibody. Theslide was counterstained with hemalum (Slide kindlyprovided by H. Merz).Result: The section showed a strong p53 signal, evenwith a very low concentration of staining antibody.G Figure 30: Detection of p53 in a human ovarianadenocarcinoma with a polyclonal antibody.A paraffin-fixed section was prepared from a partly solid,partly papillary, advanced ovarian carcinoma.
The section was stained with polyclonal sheep anti-p53 (Cat. No.1 810 928). A biotinylated donkey anti-sheep antibody(1:300 dilution, Immunotech) was used as secondaryantibody. The slide was counterstained with hemalum(Slide kindly provided by H. Merz).Result: The nuclei of the cells were strongly stained forp53.Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteins147Cell Death – Apoptosis and NecrosisApoptosis Assay MethodsDetection of apoptosis-related proteinsp53 pan ELISACat.
No. 1 828 789196 testsTypeOne-step sandwich ELISA, colorimetricUseful forQuantitation of p53 protein, both wild-type and mutant formsSamplesTissue homogenates, cell lysates, serum, or plasmaMethodLysis/homogenization of sample, followed by immunochemical determination of p53 in a microtiter plateTimeApprox.
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