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文章作者:生命科学 上传时间:2019-08-27

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

核心提示:WesternBlottingwithMonoclonalAntibodiesSample PreparationFor Protein Concentration Determin

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

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.

WesternBlottingwithMonoclonalAntibodies

Procedure

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

Sample Preparation

For Protein Concentration Determination of Cell Culture

  1. Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline .
  2. Aspirate excess PBS.
  3. Add 1ml boiling lysis buffer .
  4. Scrape cells from dish, transfer to a microcentrifuge tube, and boil for an additional 5 minutes. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle.
  5. Centrifuge the sample for 5 minutes to pellet insoluble material, then discard pellet.
  6. Dilute an aliquot of the cell lysate sample at least 10-fold for the BCA protein concentration assay .

A: Preparation of the cell lysate

Reagents

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

For Protein Gel Electrophoresis of Cell Culture

  1. Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline .
  2. Aspirate excess PBS.
  3. Add 1ml boiling 2X concentrated electrophoresis sample buffer .
  4. Scrape cells from dish, transfer to a microcentrifuge tube, and boil for an additional 5 minutes. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle. Centrifuge the sample for 10 minutes to pellet insoluble material. Discard pellet.
  5. The cell lysate sample is now ready for loading onto your gel.
  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".

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.

For Protein Concentration Determination of Whole Tissue

  1. Rapidly homogenize every 0.25g tissue in 3.5ml of boiling lysis buffer .
  2. Microwave for 10?5 seconds.
  3. Centrifuge the homogenate for 5 minutes to pellet insoluble material, then discard pellet.
  4. Dilute an aliquot of the tissue lysate sample at least 10-fold for the BCA protein concentration assay.

B : Immunoprecipitation

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. 免疫性沉淀反应。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.

Polyacrylamide Gel Electrophoresis

Guidelines for choosing the percent gel to be used for certain molecular weight proteins

4-5% gels: > 250 kDa

7.5% gels: 250-120 kDa

10% gels: 120-40 kDa

13% gels: 40-15 kDa

皇家赌场号hj85,15% gels: < 20 kDa

  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.

Time required

Afternoon and next morning.

Gel Electrophoresis

  1. If not already in electrophoresis sample buffer, add an equal volume of 2X sample buffer to all samples and boil for 3? minutes.
  2. Apply 5-20礸 total protein of cell or tissue lysate to each well of a 0.75?.0mm thick gel. For thicker gels , apply up to 25-40礸 in each well.
  3. Electrophorese until the bromophenol blue in the samples reaches the bottom of the gel. Turn off power supply. Keep gels in running buffer until ready to transfer.

C: The crosslinking of ganglioside

Protein Blotting

  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.

Wet Transfer

Note: Since extra negative charges are needed to reach 1Amp in a wet transfer system, adjust the pH of the transfer buffer to approximately pH 8.0 using NaOH.

  1. For transfer of proteins smaller than 20 kDa, transfer proteins from gel to PVDF membrane at 1Amp constant current for 45 mins or equivalent in transfer buffer .
  2. For transfer of proteins smaller than 120 kDa, transfer proteins from gel to PVDF membrane at 1Amp constant current for 1 hour or equivalent in transfer buffer .
  3. For proteins larger than 120kDa, transfer to PVDF membrane at 1Amp constant current for 90 minutes or equivalent in transfer buffer SDS .
  4. For Proteins larger than 250kDa, transfer to PVDF membrane at 1Amp constant current for 1 hour and 45 minutes or equivalent in transfer buffer SDS .

D:The detection of protein and gangliosides binding

Semi-Dry Transfer

For transfer of proteins from 10% or 13% gels to PVDF membranes semi-dry transfer can also be used. Transfer proteins to PVDF membrane at 1.2 mAmp/cm2 for 1 hour and 45 minutes in transfer buffer .

Optional

If blots are not to be used for colorimetric detection, visualize the transferred proteins by staining the membrane for 15 minutes with India ink diluted 1:1000 in wash buffer . Rinse excess stain with wash buffer before blocking.

  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.

Blocking

For All Antibodies Except Phosphotyrosine

  1. Remove the blot from the transfer apparatus or staining tray and immediately place into blocking buffer .
  2. Incubate the blot for 30 minutes at 37˚C, 1 hour at room temperature, or overnight at 4˚C.

For Phosphotyrosine Antibodies

  1. Remove the blot from the transfer apparatus or staining tray and immediately place into blocking buffer .
  2. Incubate the blot for 30 minutes at 37˚C, 1 hour at room temperature, or overnight at 4˚C.

Incubation with Primary and Secondary Antibodies

Primary antibody

  1. Dilute the antibody in the corresponding blocking buffer.
  2. Decant the blocking buffer from the blot, add the antibody solution, and incubate with agitation for 30 minutes at 37˚C, one hour at room temperature, or overnight at 4˚C.

Enzyme conjugated secondary antibody

Note: The inclusion of sodium azide is to be avoided in all steps using HRPO conjugates.

  1. Decant the primary antibody solution, add wash buffer , and wash for 30 minutes with agitation, changing the wash buffer every 3? minutes.
  2. Dilute the enzyme conjugate anti-mouse IgG:HRPO 1:2000 in wash buffer containing 5% non-fat dry milk .
  3. Decant the wash buffer, add the diluted enzyme conjugate and incubate with agitation for 30 minutes at 37˚C or one hour at room temperature.

Substrate Incubation

  1. Decant the secondary antibody solution, add wash buffer , and wash for 30 minutes with agitation, changing the wash buffer every 3? minutes.
  2. Decant wash buffer and place the blot in a plastic bag or clean tray containing chemiluminescent working solution . Rotate the bag or tray to allow the solution to cover the surface of the membrane for 1? minutes.
  3. Remove blot from the bag or tray and place it between two pieces of write-on acetate transparency film. Smooth over covered blot to remove air bubbles and excess substrate.

EXPOSE TO X-RAY FILM OR ANY SENSITIVE SCREEN. AN INITIAL EXPOSURE OF 10-60 SECONDS IS RECOMMENDED FOR FILM.

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