Antibody Protocols

Antibodies are the basis for many kinds of target-specific assays and affinity-purification techniques in biomedical and life-science research. As specific molecular probes, antibodies are integral components of nearly all research endeavors focused at the cellular or subcellular level. Cell biology is largely the result of the function and interaction of protein-mediated biochemical pathways, and consideration of these interactions at cellular and tissue levels is properly called cellular proteomics.

Antibodies are themselves proteins. Therefore, immunochemical methods are both composed of and primarily focused on proteins. Accordingly, "antibody protocols" are essentially indistinguishable from general protein methods. Thermo Scientific Pierce Protein Research Products include antibodies and other reagents to facilitate the entire range of protein research techniques.

 


Generalized Protocols

The following protocols summarize common preparative and analysis methods related to the use polyclonal and monoclonal antibodies. These procedures are intended only for use as general guides. No warranty or guarantee of performance is made or implied. Always use good laboratory practices and handle all materials with care.

 


 

Antibody Neutralization with Peptide

  1. Reconstitute lyophilized peptide with 0.1 ml distilled water or PBS. Be careful to reconstitute entire contents of vial as some product may be on the lid.
  2. To a separate tube add appropriate amount of antibody.
  3. To that tube add an equal weight of peptide per volume antibody (i.e. 1 ug peptide/1 ul antibody).
  4. Dilute the antibody/peptide solution to approximately 1.0 ml with Western blot blocking buffer and vortex well.
  5. Incubate at room temperature 15 to 30 minutes.
  6. Use antibody for procedures as you would normally.

Antibody Storage

For more information, see our list of Tech Tips and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products. In particular, see Tech Tip #43: Protein Stability and Storage.

Antigen Retrieval (EDTA) Protocol

  1. Place slides or specimens into rack or other suitable container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
  2. Incubate the specimens for 10 minutes at room temp in 1.0 mM EDTA (pH 8.0, adjust with sodium hydroxide) with occasional agitation.
  3. Move the entire container into the microwave oven. Mark the height of the buffer.
  4. Microwave the sample at 750 W for three cycles of 5 min. each. After each cycle, replenish any lost liquid from the slide container by the addition of distilled water to the original marked height of the buffer.
  5. Remove the container and allow it to cool to room temperature.
  6. Wash specimens twice with PBS.

Antigen Retrieval (Pressure Cooker) Protocol

  1. Place slides or specimens into rack or other suitable container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
  2. Incubate the specimens for 10 minutes at room temp in 1.0 mM EDTA (pH 8.0, adjust with sodium hydroxide) with occasional agitation.
  3. Fill a stainless steel pressure cooker approximately one-third full of above EDTA solution. On hotplate, heat until boiling.
  4. Load slides into the buffer and seal the pressure cooker.
  5. Allow pressure to reach maximum. Incubate samples for two minutes at maximum.
  6. Remove cooker from heat and cool by placing it under cold running water.
  7. Open cooker and remove slides. Wash specimens twice with PBS.

Antigen Retrieveal (Protease Treatment) Protocol

  1. Place slides or specimens into rack or other suitable container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
  2. Incubate the specimens for 10 minutes at room temp in PBS with occasional agitation.
  3. Pour off the PBS and incubate the specimen in a 0.1% trypsin, 0.1% calcium chloride, 20 mM Tris (pH 7.8) solution for 2-20 minutes at room temperature.
  4. Stop the digestion by gently rinsing the specimen under the cold tap for 5 minutes.
  5. Samples are now ready for the addition of antibody.

ELISA Protocol

  1. Bind Antigen to Microplate
    1. Dispense 1-5 ug antigen in PBS into each well.
    2. Cover the plate with parafilm and place in the refrigerator overnight.
    3. Remove and discard antigen solution from plate.
  2. Blocking and Primary Antibody Incubation
    1. Block each well in the entire microplate by adding 200 ul of 2.5 mg/ml BSA in PBS to each well.
    2. Allow plate to stand at room temperature for 2 hours.
    3. Remove and discard blocking solution from plate.
    4. Add the appropriate amount of primary antibody to each well and incubate at room temperature for 1 hour.
    5. Remove and discard incubation solution from plate.
    6. Wash each well using TBST (TBS containing 0.1% Tween 20). Do NOT cross-contaminate wells.
    7. Repeat washing step for a total of 5 washes.
  3. Secondary Incubation and Detection
    1. Add appropriate amount of secondary antibody solution to wells and incubate at room temperature for 1 hour.
    2. Remove and discard secondary antibody solution from plate.
    3. Wash each well using TBST (TBS containing 0.1% Tween 20). Do NOT cross-contaminate wells.
    4. Repeat washing step for a total of 5 washes.
    5. Add the appropriate detection reagent, incubate, and stop the color development according to manufacturer's recommendations.
    6. Read the plate according to manufacturer's recommendations.

For more information, see our Overview of ELISA and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.

Immunocytochemistry/Immunofluorescence Protocol

  1. Fixation
    1. Fix freshly washed cells with ice-cold ethanol, methanol, or paraformaldehyde for 1-10 minutes at room temperature.
    2. Wash slides twice with ice cold PBS for 5 minutes.
  2. Blocking and Primary Antibody Incubation
    1. Block cells with 10% serum from the species from which the secondary antibody was taken or 8% BSA.
    2. Incubate for 1 hour at room temperature in a humidified chamber.
    3. Wash samples twice with PBS for 5 minutes each.
    4. Incubate cells in a humidified chamber with primary antibody solution (primary antibody in PBS containing 2% BSA) for 1-4 hours at room temperature.
    5. Wash three times with PBS containing 2% BSA for 5 minutes.
  3. Secondary Antibody Incubation and Detection
    1. Incubate slides in a humidified chamber with secondary antibody solution (primary antibody in PBS containing 2% BSA) according to manufacturer's protocols.
    2. Wash three times with PBS containing 2% BSA for 5 minutes each.
    3. Detect according to manufacturer's protocols.

Immunohistochemistry Protocol (Frozen Sections)

  1. Fixation and Sectioning
    1. Freeze and section tissue samples.
    2. Fix sections in Bouin's solution, -20°C acetone, -20°C methanol, or formaldehyde for 2-30 minutes. (NOTE: Fixation time will depend on species, tissue origin and size of tissue sections).
  2. Blocking and Primary Antibody Incubation
    1. Block slides with 10% serum from the species of the secondary antibody or 8% BSA.
    2. Incubate for 30 minutes to 1 hour at room temperature in a humidified chamber.
    3. Wash samples in PBS containing 2% BSA for 5 minutes.
    4. Incubate slides in a humidified chamber overnight with primary antibody solution (primary antibody in PBS containing 2% BSA).
    5. Wash three times with PBS containing 2% BSA for 5 minutes.
  3. Secondary Antibody Incubation and Detection
    1. Incubate slides in a humidified chamber with secondary antibody solution (secondary antibody in PBS containing 2% BSA) according to manufacturer's protocols.
    2. Wash three times with PBS containing 2% BSA for 5 minutes.
    3. Detect according to manufacturer's protocols.

Immunohistochemistry Protocol (Paraffin Sections)

  1. Fixation and Sectioning
    1. Fix dissected tissues in 2% paraformaldehyde, Bouin's solution, or other fixative for 30 minutes to overnight. (NOTE: Fixation time will depend on species, tissue origin and size of tissue sections).
    2. Embed tissue in paraffin.
    3. Section the tissues 5-10 micrometers thick.
    4. If using paraformaldehyde as fixative antigen retrieval may be necessary.
  2. Deparaffinization and Tissue Rehydration
    1. Deparaffin samples by incubating sections 2-3 times in xylene for 10 minutes each.
    2. Hydrate samples by placing in 100% ethanol for 2 x 3 minutes each, then in 95%, 70%, 50%, 30% ethanol for 1 x 2 minutes each.
  3. Blocking and Primary Antibody Incubation
    1. Block slides with 10% serum from the species from which the secondary antibody was taken or 8% BSA.
    2. Incubate for 30 minutes to 1 hour at room temperature in a humidified chamber.
    3. Wash samples in PBS containing 2% BSA for 5 minutes.
    4. Incubate slides in a humidified chamber overnight with primary antibody solution (primary antibody in PBS containing 2% BSA).
    5. Wash three times with PBS containing 2% BSA for 5 minutes.
  4. Secondary Antibody Incubation and Detection
    1. Incubate slides in a humidified chamber with secondary antibody solution (secondary antibody in PBS containing 2% BSA) according to manufacturer's protocols.
    2. Wash three times with PBS containing 2% BSA for 5 minutes.
    3. Detect according to manufacturer's protocols.

Immunoprecipitation Protocol

  1. Sample Preclearing
    1. Wash protein A- or G-Sepharose or protein L-Agarose with 10X volume of RIPA buffer.
    2. Vortex and centrifuge for 1 minute in a microfuge.
    3. Resuspend the pellet in the original volume that the protein A or protein G matrix was in.
    4. Add protein A- or G-Sepharose or protein L-Agarose to the sample and incubate at 4°C for 30 minutes with shaking.
    5. Centrifuge for 1 minute in a microfuge to pellet absorbed nonspecific proteins and insoluble material. Discard.
  2. Incubation with Primary Antibody
    1. Add primary antibody to precleared sample and incubate for 1 hour at 4°C with gentle agitation.
    2. Add protein-A or -G Sepharose beads and incubate 1 hour at 4° C with gentle agitation.
    3. Centrifuge for 1 minute in a microfuge. Wash the pellet in 1 ml of RIPA buffer.
    4. Repeat twice.
  3. Removal of Antibody-antigen Complex
    1. Resuspend immunoprecipitate in a buffered solution with either 1% SDS and 15 mM beta-mercaptoethanol or 8M urea.
    2. Heat at 90-100°C for 5-20 minutes with occasional vortexing.
    3. Centrifuge for 1-2 minutes in a microfuge.
    4. The supernatant is ready to be analyzed by Western blot.

For more information, see our Overview of Immunoprecipitation and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.

Protein Extraction Protocols

Note: Keep equipment cold and everything else on ice!

  1. Prepare Protein Extraction Buffer (EB)
    1. 20 mM Tris-HCl, pH 7.4
    2. 10 mM NaCl
    3. 10 mM KCl
    4. 3 mM MgCl2
    5. 1 mg/ml protease inhibitors (leupeptin, antipain, aprotinin, pepstatin, etc.)
  2. Protein Extraction from Cultured Cells
    1. Rinse cells in ice-cold PBS.
    2. Scrape cells (~3-5 mls/T75 flask or less) in PBS, centrifuge ~1300 x g for 5 minutes, remove PBS.
    3. Gently resuspend cells in EB.
    4. Add Nonidet P-40 Detergent to 0.5%.
    5. Dounce homogenize (10-20 passes with the "B" or "LOOSE" pestle).
    6. Centrifuge at ~2000 x g for 20 minutes (Pellet consists of cell nuclei-use these for isolation of DNA or nuclear proteins).
    7. Aliquot supernatant (100-500ml depending on starting volume) and freeze in liquid nitrogen. Store at -80°C.
    8. Quantitate protein (OD280 1 = 1.0 mg/ml).
  3. Protein Extraction from Tissue (fresh or frozen)
    1. Mince tissue then rinse several times in PBS prior to flash freezing and/or homogenizing. Use mortar and pestle, if available, to powder frozen tissue.
    2. Place minced or powdered tissue in tissue homogenizer.
    3. Add EB (~5 ml/gm tissue).
    4. Add 50 ul Anti-Foam/ml of EB (and more as needed to prevent frothing and foaming).
    5. Homogenize for several minutes on high speed (little or no tissue chunks should remain).
    6. Add Nonidet P-40 Detergent to 0.5% and rock in cold 10 - 20 minutes.
    7. Dounce homogenize (10-20 passes with the "B" or "LOOSE" pestle).
    8. Centrifuge homogenate at 10,000 x g for 10-15 minutes at 4°C to remove particulate debris.
    9. Remove supernatant (SN) avoiding insoluble material in pellet and upper, lipid-containing phase (especially in whole brain homogenate) if present.
    10. For microsome preps, centrifuge SN at 100,000 x g for 60 minutes at 4°C. Pellet is crude microsomal fraction and may require additional washing steps to produce a cleaner prep.
    11. Filter SN through low-protein-binding membrane.
    12. Aliquot as above, flash-freeze, and store at -80°C.
    13. Quantitate protein (OD280 1 = 1.0 mg/ml).

For more information, see our Overview of Cell Lysis and Protein Extraction and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.



Reconstitution of Lyophilized Peptide

Generally, lyophilized peptides may be reconstituted using sterile H2O in a volume that will restore the peptide to the desired concentration. Ideally, oxygen-free-water should be used to avoid oxidation of cysteine, methionine, and tryptophan residues. Alternatively, an aqueous acetic acid solution (up to 50%) may be used to dissolve peptides with strong hydrophobic properties.

Subcellular Fractionation Protocol

  1. Homogenize tissue. (Different tissues require different homogenization techniques and optimization is required.)
  2. 1,000 x g for 10 min. (pellets nuclei, heavy mitochondria, plasma membrane sheets)
  3. 3,000 x g for 10 min. (heavy mitochondria, plasma membrane fragments)
  4. 6,000 x g for 10 min. (mitochondria, lysosomes, peroxisomes, intact Golgi)
  5. 10,000 x g for 10 min. (mitochondria, lysosomes, peroxisomes, intact Golgi membranes)
  6. 20,000 x g for 10 min. (lysosomes, peroxisomes, Golgi membranes, large dense vesicles [e.g., rough ER])
  7. 100,000 x g for 10 min. (all vesicles from ER, plasma membrane, Golgi, endosomes, etc.)

For more information, see our Overview of Cell Fractionation and Organelle Isolation and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.

Western Blot Protocol

  1. Polyacrylamide gel electrophoresis and blotting
    1. Add an appropriate amount of electrophoresis sample buffer (1X = 125mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% beta-mercaptoethanol) to all samples.
    2. Heat to 95°C for 3-5 minutes.
    3. Load 5-100 ug total protein in a volume that is appropriate for the size of the wells.
    4. Electrophorese proteins for the appropriate time according to the manufacturers instructions.
    5. Transfer proteins from the gel to a suitable membrane (e.g. nitrocellulose, PVDF, etc.) following the manufacturers protocol for transfer.
    6. High molecular weight proteins (>120 kDa) can be wet transferred more efficiently if transfer time is increased and 0.05% SDS is included in the transfer buffer.
  2. Blocking
    1. Remove the filter from the transfer apparatus and rinse in PBST/TBST to remove loose acrylamide.
    2. Transferred proteins can be visualized by staining the membrane for 15-30 minutes with Ponceau S.
    3. Remove stain from filter by washing with PBST/TBST.
    4. Place filter into blocking solution.
    5. Block for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.
  3. Incubation with primary antibody
    1. Decant the blocking buffer and add the antibody, diluted in blocking buffer as suggested in the product description sheet.
    2. Incubate with agitation for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.
  4. Incubation with secondary antibody
    1. Wash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1% Tween 20), changing the wash buffer every 5 minutes.
    2. Decant the wash solution and add AffiniClear HRP-conjugated secondary antibody, diluted in 5% non-fat dry milk in wash buffer.
    3. Incubate for 30 minutes at 37°C, 1 hour at room temperature, or overnight at 4°C.
    4. Decant the antibody conjugate and wash for 30 minutes with agitation in wash buffer (TBS or PBS with 0.1% Tween 20), changing the wash buffer every 5 minutes.
  5. Substrate incubation (ECL)
    1. Decant washing buffer and place the blot in a plastic bag or clean tray containing the development working solution (0.125 ml/cm2) for 1-5 minutes.
    2. Agitate bag or tray to cover the surface of the membrane.
    3. Remove the blot from the bag or tray and place it between two pieces of write-on transparency film.
    4. Smooth over the covered blot to remove air bubbles and excess substrate.
    5. Expose to X-ray film or any sensitive screen. (Check manufacturer's instructions for specific ECL reagents and procedures.)

For more information, see our Overview of Western Blotting and related articles in our Protein Methods Library for Thermo Scientific Pierce Protein Research Products.

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