Skip to content

Basket

You currently have no items in your basket.

Total (excl. vat) £0.00
View basket & checkout

Technical Recommendations for Monoclonal Antibody Use

Our monoclonal antibodies are suitable for use in a wide range of standard immunochemical applications and are intended for use as primary antibodies. Antibody reactivity may vary depending on the biological origin of the sample, sample preparation procedures, and specific assay conditions. The following recommendations provide general guidance for the use of InVivo monoclonal antibodies in immunochemical assays. Users are advised to empirically determine optimal conditions within their own experimental protocols.

For immunochemical detection, secondary antibodies conjugated to enzymes or other reporter molecules are commonly employed. Users should carefully consult the antibody isotype information to ensure appropriate selection of secondary antibodies. The use of secondary antibody conjugates specific for mouse IgG or IgM is recommended, with reagents selected to minimize cross-reactivity with immunoglobulins from other species.

General Information

Monoclonal antibodies are typically supplied in phosphate-buffered saline (PBS; pH 7.4) without additional stabilizing agents. Standard reaction buffers, including Tris-based buffers (e.g. Tris or TBS), phosphate-buffered saline (PBS), carbonate buffer, or equivalent buffer systems are generally suitable for use with monoclonal antibodies. Buffer systems are typically applied at concentrations of 10-50 mM and within a pH range commonly used for immunochemical assays (approximately pH 7.2-8.5).

The antibodies are compatible with reaction and washing buffers containing non-ionic detergents such as Tween® 20 or Triton™ X-100 at concentrations typically ranging from 0.05–0.2%. For blocking or stabilization purposes, bovine serum albumin (BSA) is commonly used in immunoprecipitation and ELISA-based assays, while skim milk powder is frequently applied in Western blotting, typically at concentrations of 1–5%. If specific buffer compositions or conditions are required, users should refer to the individual monoclonal antibody datasheet for detailed recommendations

Immunohistochemistry

  1. Tissue sections fixed in 4.5% formaldehyde are recommended. Alternative fixation methods may be suitable depending on tissue type and target epitope
  2. Incubate slides in citrate buffer at 95 °C for 20 min. Rinse with water, then incubate in concentrated formic acid at room temperature for 1 min. Subsequently, wash slides thoroughly with water or antibody dilution buffer. Alternative antigen retrieval methods may be applied as appropriate.
  3. Dilute the primary antibody to a final concentration of 0.1–2 µg/mL in PBS (pH 7.4) containing 3% bovine serum albumin (BSA). Incubate tissue sections at room temperature for 30 min. Wash slides three times with TBS (pH 7.0-7.5).
  4. Incubate with an HRP-conjugated secondary antibody diluted according to the manufacturer’s instructions for use (IFU) at room temperature for 30 min.
  5. Wash slides three times with TBS (pH 7.0-7.5). 6. Develop staining using DAB according to the manufacturer’s IFU.

Western Blot 

  1. Block the membrane containing the transferred antigen using 5% skim milk powder in Tris-based buffer (pH 9.0-10.0) supplemented with 0.1% Triton™ X-100. Incubate at room temperature for 1 h.
  2. Dilute the monoclonal antibody to a final concentration of 1–2 µg/mL in blocking buffer and incubate the membrane overnight at room temperature or at 2-8 °C.
  3. Wash the membrane three times with Tris-based buffer containing 0.1% Triton™ X-100.
  4. Dilute the HRP-conjugated anti-mouse Ig secondary antibody according to the manufacturer’s instructions for use (IFU) in blocking buffer. Incubate the membrane at room temperature for 1–2 h.
  5. Wash the membrane five times with Tris-based buffer containing 0.1% Triton™ X-100. 6. Incubate the membrane with the appropriate detection solution at room temperature and terminate the reaction under visual control by rinsing with water.

Immunoprecipitation

Bead Preparation (Magnetic Beads: DynaBeads™ M-280 Streptavidin)

  1. Vortex the bead suspension thoroughly. Transfer 100 µL of beads into a 1.5 mL microcentrifuge tube.
  2. Place the tube on a magnetic rack for 3 min to collect the beads and carefully discard the supernatant.
  3. Wash the beads three times with 500 µL PBS (pH 7.4) containing 3% bovine serum albumin (BSA) and 0.05% Tween® 20. For each wash, gently resuspend the beads by pipetting, collect using the magnetic rack for 3 min, and remove the supernatant.

Coating of Beads with Monoclonal Antibody 

  1. Add 20 µg of biotin-conjugated monoclonal antibody, diluted in PBS (pH 7.4) containing 3% BSA and 0.05% Tween® 20, to 1 mg of DynaBeads™ M-280 Streptavidin.
  2. Incubate at room temperature for 30 min with gentle agitation.
  3. Wash the antibody-coated beads three times with 500 µL PBS (pH 7.4) containing 3% BSA and 0.05% Tween® 20, as described above.
  4. Collect the beads using a magnetic rack for 3 min and discard the supernatant.
  5. Perform an additional three washes with the same buffer.
  6. Aliquot the coated beads at 0.2 mg per tube. The aliquots may be stored at 2–10 °C until use.

Immunoprecipitation of Antigen

  1. Add 100–1000 µL of sample (undiluted or diluted in PBS pH 7.4 containing 3% BSA and 0.05% Tween® 20) to one aliquot (0.2 mg) of monoclonal antibody–coated beads.
  2. Incubate overnight at 2–10 °C with continuous rotation.
  3. Collect the beads using a magnetic rack for 3 min and discard the supernatant.
  4. Wash the beads three times with 500 µL PBS (pH 7.4) containing 3% BSA and 0.05% Tween® 20, as described above.
  5. Successful immunoprecipitation can be confirmed by analysing the captured antigen via SDS-PAGE followed by Western blotting.


Important information: All InVivo catalogue products including the monoclonal antibodies are intended for research use only and are designed for use in in vitro immunochemical applications, including immunohistochemistry, Western blotting, and immunoprecipitation. They are not intended for use in diagnostic, therapeutic, or in vivo applications. Experimental conditions may require optimization depending on the specific target antigen, sample type, and assay format.