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新陈代谢标记与免疫沉淀,免疫沉淀反应

文章作者:生命科学 上传时间:2019-08-27

核心提示:Purpose and backgroundsAbout nuclide...35-SDecay: Half life: 87.5 daysThick acrylic shield can Purpose and backgroundsAbout nuclide...35-SDecay: Half life: 87.5 days

核心提示:Metaboliclabeling&ImmunoprecipitationPurpose and backgroundsAbout nuclide...35-SDecay: Half life: 87.5 days

核心提示:OutlineImmunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has

Thick acrylic shield can block most of the emission. Because of the low energy, it is impossible todetect spillage/contamination by usual survey meter. Wipe test LSC is necessary.

Thick acrylic shield can block most of the emission. Because of the low energy, it is impossible todetect spillage/contamination by usual survey meter. Wipe test LSC is necessary.

Outline

Immunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has been raised. This primary antibody is either already bound to agarose or can be bound to the protein A/agarose beads during the procedure in order to physically separate the antibody-antigen complex from the remaining sample.

Chemical properties...Use either 35S-methionine or cysteine or both. Recommend using Expre35S35S Protein labeling mix . Before opening the cap of the vial, thaw the solution completely and open the cap in the hood so that any vaporized materials can be trapped in the hood.

Chemical properties...Use either 35S-methionine or cysteine or both. Recommend using Expre35S35S Protein labeling mix . Before opening the cap of the vial, thaw the solution completely and open the cap in the hood so that any vaporized materials can be trapped in the hood.

Supplies / Equipment

  • sterile 1.5 µl microfuge tubes
  • sterile pipette tips
  • micropipettes
  • waterbath 100°C
  • microfuge 4°C
  • rocking platform 4°C
  • rocking platform 25°C / room temperature

Principle of radiolabelingCells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling , and additional incubation with excess concentration of unlabeled Met Cys is needed for complex glycoproteins like integrins to get expressed as a maturated form. Typically, overnight chase is recommended for metabolic labeling of fully processed form of integrins, but optimum time may vary depending on each protein.

Principle of radiolabelingCells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling , and additional incubation with excess concentration of unlabeled Met Cys is needed for complex glycoproteins like integrins to get expressed as a maturated form. Typically, overnight chase is recommended for metabolic labeling of fully processed form of integrins, but optimum time may vary depending on each protein.

皇家赌场号hj85,Reagents

  • Immunoprecipitation dilution buffer
  • Immunoprecipitation wash buffer
  • Immunoprecipitation buffer
  • Electrophoresis sample buffer
  • Antisera 0.5 - 5 µl, antibody-agarose conjugates
  • 10% SDS

Materials

Materials

Procedure 1

  1. Preparation of cell lysate
    1. Rinse the cells on a confluent 60mm culture dish with PBS,
    2. Lyse with the addition of 0.5ml boiling 1% SDS, 1.0mM sodium vanadate, 10mM Tris-HCl pH 7.4.
    3. Transfer lysate to a 1.5ml microcentrifuge tube and boil for an additional 5 minutes.
    4. Sonicate briefly or pass several times through a 26 gauge needle and centrifuge for 5 minutes. This is a "total cell lysate ".
  2. Preparation of cell lysate
    1. Rinse the cells in a confluent 60 mm culture dish with PBS, then lyse the cells with the addition of 0.5 ml cold immunoprecipitation buffer , maintaining constant agitation for 30 minutes at 4°C.
    2. Scrape the cells from the dish and pass several times through a 26 gauge needle to disperse any large aggregates.
    3. Remove insoluble materials by centrifuging the cell lysates for 15 minutes at 4°C in a microcntrifuge. The supernatant is the "total cell lysate ".
  3. Immunoprecipitation with soluble antibodies
    1. To a microcentrifuge tube, add 1-5 µg polyclonal or monoclonal antibody, 400 µl dH2O, 500 µl of 2 x immunoprecipitation buffer, and 100 µl total lysate containing approximately 200 - 500 µg total protein.
    2. Vortex and incubate at 4°C for 1 hour. .
    3. Add 50 µl 10 % protein A or protein A-sepharose. Vortex and incubate with agitation for 30 minutes at 4°C.
    4. Wash 3 x by centrifugation with 1 x immunoprecipitation buffer.
    5. Resuspend the pellet in 30 µl of 2 x concentrated electrophoresis sample buffer, boil for 5 min., the centrifuge for 5 min.
    6. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.
  4. Immunoprecipitation with Antibody-Agarose Conjugates
    1. To a microcentrifuge tube, add 25µl of a 50% suspension of antibody-agarose conjugate, 400µl H2O, 500µl 2X immunoprecipitation buffer, and 100µl total lysate containing 200-500µg total protein.
    2. Vortex and incubate at 4°C for 1 hour.
    3. Wash with 1X immunoprecipitation buffer by centrifuging for 4 minutes in a microcentrifuge. Repeat wash.
    4. Resuspend the pellet in 30µl 2X concentrated electrophoresis sample buffer, boil for 5 minutes, then centrifuge for 5 minutes.
    5. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.

•Complete medium: DMEM supplmented w/ 10% FCS, NEAA, Na-pyruvate, and Gln•Expre35S35S Protein labeling mix , 7.9mCi/ml• Transfection reagents: CaCl2, x2HBS, DNA•Labeling Medium:RPMI1640 supplemented w/ 10% dialyzed FCS, & Gln• Chase Medium:Labeling medium 500µg/ml Cysteine-HCl, 100µg/ml methionine, sterile filtered• Tris-buffered saline : 20mM Tris, 150mM NaCl, pH 7.4• X1000 Protease inhibitor soln: 500mM PMSF in EtOH, 10mg/ml leupeptin in water, 10mg/ml pepstatin A in dimethylformamide• IP buffer: 1% Triton x-100, 0.05% NP-40 in TBS• x2 IP buffer: 2% Triton x-100, 0.1% NP-40 in TBS• ProteinG agarose • Gel fixation solution: 7% acetic acid, 25% MeOH in water

•Complete medium: DMEM supplmented w/ 10% FCS, NEAA, Na-pyruvate, and Gln•Expre35S35S Protein labeling mix , 7.9mCi/ml• Transfection reagents: CaCl2, x2HBS, DNA•Labeling Medium:RPMI1640 supplemented w/ 10% dialyzed FCS, & Gln• Chase Medium:Labeling medium 500µg/ml Cysteine-HCl, 100µg/ml methionine, sterile filtered• Tris-buffered saline : 20mM Tris, 150mM NaCl, pH 7.4• X1000 Protease inhibitor soln: 500mM PMSF in EtOH, 10mg/ml leupeptin in water, 10mg/ml pepstatin A in dimethylformamide• IP buffer: 1% Triton x-100, 0.05% NP-40 in TBS• x2 IP buffer: 2% Triton x-100, 0.1% NP-40 in TBS• ProteinG agarose • Gel fixation solution: 7% acetic acid, 25% MeOH in water

Procedure 2

Immunoprecipitation of a serum sample

  1. Add 1/10 vol. of 10% SDS to sample. Vortex and heat for 4 min. at 100°C.
  2. Immediately add 4 volumes of immunoprecipitation dilution buffer. Vortex and spin 5 min. in microfuge.
  3. Transfer to a new tube, avoiding pellet.
  4. To a microfuge tube, add 1-5 µg of primary antibody. Place on 4 °C rocker overnight.
  5. If primary antibody was not already conjugated with agarose, add 20 µl protein A-sepharose per tube.
  6. Place on 25°C rocker for 90 min., spin down pellet .
  7. Wash pellet 3 times with detergent wash buffer, then once with non-detergent wash buffer.
  8. Add treatment buffer. Add 1/20 volume 2-mercaptoethanol.
  9. Boil samples 4 min. Spin down pellet. Remove and save supernatant, this is the sample.

• Gel soaking solution: 1% glycerol, 5% PEG 8000 in water

• Gel soaking solution: 1% glycerol, 5% PEG 8000 in water

Time required

Afternoon and next morning.

Procedure

Procedure

Transfection of 293 cells Prepare transfected cells in 6-well plate. Use 2µg DNA/subunit/well. After 7-9h incubation with DNA:calcium phosphate, rinse once and replace with fresh medium .Metabolic labeling1. Transfected cells -> 24h after medium change, rinse cells twice with Labeling Medium.2. Add 1.5ml/well Labeling Medium3. Add 20-30µl of [35S] methionine/cysteine /well4. Culture 1h at 37°C 5. Add 1.5ml/well Chase Medium6. Culture o/n 7. Harvest metabolically labeled cells *Immunoprecipitationi) preparation of cell lysate 1. After removing the medium, detach cells by suspending in TBS, transfer to microfuge tube2. Spin down & resuspend in 0.5 ml TBS3. Add 1µl each of x1000 protease inhibitor soln4. Solubilize cells by adding 0.5 ml x2 IP buffer5. on ice for 20min6. Centrifuge at maximum speed for 15min7. Save supernatant ii) preparation of culture supernatant *1. Harvest medium

Transfection of 293 cells Prepare transfected cells in 6-well plate. Use 2µg DNA/subunit/well. After 7-9h incubation with DNA:calcium phosphate, rinse once and replace with fresh medium .Metabolic labeling1. Transfected cells -> 24h after medium change, rinse cells twice with Labeling Medium.2. Add 1.5ml/well Labeling Medium3. Add 20-30µl of [35S] methionine/cysteine /well4. Culture 1h at 37°C 5. Add 1.5ml/well Chase Medium6. Culture o/n 7. Harvest metabolically labeled cells *Immunoprecipitationi) preparation of cell lysate 1. After removing the medium, detach cells by suspending in TBS, transfer to microfuge tube2. Spin down & resuspend in 0.5 ml TBS3. Add 1µl each of x1000 protease inhibitor soln4. Solubilize cells by adding 0.5 ml x2 IP buffer5. on ice for 20min6. Centrifuge at maximum speed for 15min7. Save supernatant ii) preparation of culture supernatant *1. Harvest medium

  1. Spin down to remove cell debries3. Adjust pH by adding 50µl 1M Tris pH 8.04. Save supernatant
  1. Spin down to remove cell debries3. Adjust pH by adding 50µl 1M Tris pH 8.04. Save supernatant

iii) IP1. Prepare 1.7ml Eppendorf tubes 2. Add 100-400µl of 35S-labeled culture sup or cell lysate3. Add mAb 4. Add 20µl of ProteinG-agarose >>>use wide-hole tips for transferring gel suspension

iii) IP1. Prepare 1.7ml Eppendorf tubes 2. Add 100-400µl of 35S-labeled culture sup or cell lysate3. Add mAb 4. Add 20µl of ProteinG-agarose >>>use wide-hole tips for transferring gel suspension

  1. Adjust total vol to 500µl with TBS or IP buffer 6. Incubate at 4°C for 1-3h7. Spin down @ 6000rpm for 3min 8. Remove supernatant 9. Resuspend in 500µl of TBS and spin down

  2. Remove final wash solution roughly with P1000 pipet, then completely with narrow-mouth, gel-loading tip attached to P200 pipet so that you won’t take out ProteinG beads.11. To the slightly moist pellet of beads,add 30µl SDS-PAGE sample buffer 12. Wait for 5min, then boil for 1min13. Spin down, and load 5-10µl onto SDS-PAGE gel14. After electrophoresis, fix gel in fixation soln. for 30min15. Remove solution and put gels into enhancer solution for 1h16. Transfer EN3HANCE solution to waste bottle, soak gels into soaking solution 17. Dry gels18. Autoradiography Key wordsTransfection, 293 cells, 35-S, metabolic labeling, immunoprecipitation, autoradiography, monoclonal antibody

  1. Adjust total vol to 500µl with TBS or IP buffer 6. Incubate at 4°C for 1-3h7. Spin down @ 6000rpm for 3min 8. Remove supernatant 9. Resuspend in 500µl of TBS and spin down

  2. Remove final wash solution roughly with P1000 pipet, then completely with narrow-mouth, gel-loading tip attached to P200 pipet so that you won’t take out ProteinG beads.11. To the slightly moist pellet of beads,add 30µl SDS-PAGE sample buffer 12. Wait for 5min, then boil for 1min13. Spin down, and load 5-10µl onto SDS-PAGE gel14. After electrophoresis, fix gel in fixation soln. for 30min15. Remove solution and put gels into enhancer solution for 1h16. Transfer EN3HANCE solution to waste bottle, soak gels into soaking solution 17. Dry gels18. Autoradiography Key wordsTransfection, 293 cells, 35-S, metabolic labeling, immunoprecipitation, autoradiography, monoclonal antibody

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