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免疫性沉淀反应,糖脂绑定剖判【皇家赌场号h

文章作者:生命科学 上传时间:2019-08-27

核心提示:ProcedureA: Preparation of the cell lysateRinse a 60 mm culture dish of confluent cells with 10 mM Tris

核心提示:ProcedureA: Preparation of the cell lysateRinse a 60 mm culture dish of confluent cells with 10 mM Tris

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

Procedure

Procedure

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.

A: Preparation of the cell lysate

A: Preparation of the cell lysate

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
  1. Rinse a 60 mm culture dish of confluent cells with 10 mM Tris-HCl,PH 7.4, 0.15 M NaCl, 1 mM MnCl2 ,and 0.2 mM PMSF.
  2. Lyse the cells with 0.5ml cold buffer
  3. Maintain constant agitation for 20 minutes at 4 oC.
  4. Scrape the cells from the dish and centrifuge for 15 minutes,the supernatant is the "total cell lysate".
  1. Rinse a 60 mm culture dish of confluent cells with 10 mM Tris-HCl,PH 7.4, 0.15 M NaCl, 1 mM MnCl2 ,and 0.2 mM PMSF.
  2. Lyse the cells with 0.5ml cold buffer
  3. Maintain constant agitation for 20 minutes at 4 oC.
  4. Scrape the cells from the dish and centrifuge for 15 minutes,the supernatant is the "total cell lysate".

Reagents

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

B : Immunoprecipitation

B : Immunoprecipitation

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.
  1. Add 4 µg of antibody,400 µl of H2O,400 µg total protein to microcentrifuge tube.
  2. Vortex and incubate at 4celsius degree for 1 Hr.
  3. Add 10 µl 50% protein A : Agrose, vortex and incubate for 30 minutes at 4 oC.
  4. Centrifuge the agarose solution for 5 minutes and discard the supernatant.
  5. Wash with lysis buffer,by centrifuging 5 minutes , repeat wash twice.
  1. Add 4 µg of antibody,400 µl of H2O,400 µg total protein to microcentrifuge tube.
  2. Vortex and incubate at 4celsius degree for 1 Hr.
  3. Add 10 µl 50% protein A : Agrose, vortex and incubate for 30 minutes at 4 oC.
  4. Centrifuge the agarose solution for 5 minutes and discard the supernatant.
  5. Wash with lysis buffer,by centrifuging 5 minutes , repeat wash twice.

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.

C: The crosslinking of ganglioside

C: The crosslinking of ganglioside

Time required

Afternoon and next morning.

  1. Add 200-400 µl 50-100 uM ganglioside to microcentrifuge tube.
  2. Add 2.5x 10,000 particles of 1 uM Fluosphere beads.
  3. Mix overnight at 4 oC with 200 µl 5mg/ml 1-ethyl-3--carbodiimide.
  4. Wash with PBS, by centrifuging,repeat wash 3 times.
  5. Resuspend the bead in PBS solution.
  1. Add 200-400 µl 50-100 uM ganglioside to microcentrifuge tube.
  2. Add 2.5x 10,000 particles of 1 uM Fluosphere beads.
  3. Mix overnight at 4 oC with 200 µl 5mg/ml 1-ethyl-3--carbodiimide.
  4. Wash with PBS, by centrifuging,repeat wash 3 times.
  5. Resuspend the bead in PBS solution.

皇家赌场号hj85,D:The detection of protein and gangliosides binding

D:The detection of protein and gangliosides binding

  1. Add 5-10 µl Fluoro-bead to microcentrifuge tube with immunoprecipitated protein.
  2. incubate 1 Hr at room temperature.
  3. Wash with PBS or lysis buffer for 3 times.
  4. Monitor by Fluoro-microscope.
  1. Add 5-10 µl Fluoro-bead to microcentrifuge tube with immunoprecipitated protein.
  2. incubate 1 Hr at room temperature.
  3. Wash with PBS or lysis buffer for 3 times.
  4. Monitor by Fluoro-microscope.

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