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Immunostaining Protocols

Additional Information

Standard Immunostaining Protocol (5.75 hours)

  1. If membrane was dried and stored at 4 °C, re-wet in 100% methanol for a few seconds. Discard methanol and rinse membrane in deionized water.
  2. Block unoccupied protein binding sites on membrane by placing membrane in Blocking Buffer. Incubate on rocker/shaker at room temperature for 1-2 hours.
  3. Pour off the blocking solution and add 20 mL of 1° antibody, diluted according to manufacturer's recommendations in Diluent Buffer, to membrane. Incubate on rocker/shaker at room temperature for 2-3 hours.
  4. Pour off 1° antibody and wash membrane for 5 minutes in TTBS. Repeat wash two additional times.
  5. Incubate blots with 20 mL of 2° antibody diluted 1:8,000 - 1:50,000 (or manufacturer's recommended dilution) in Diluent Buffer. Incubate on rocker/shaker at room temperature for 1 hour.
  6. Wash membrane for 5 minutes in TTBS and incubate with 20 mL of AP reagent diluted 1:1000 (or manufacturer's recommended dilution) in Diluent Buffer. Incubate on rocker/shaker at room temperature for 1 hour.
  7. Wash membrane for 5 minutes in TTBS.
  8. Prepare Substrate Development Solution by adding 132 µL of NBT stock and 66 µL of BCIP stock to 20 mL of AP Substrate Buffer. Do not allow membrane to dry while preparing Substrate Development Solution.
  9. Add Substrate Development Solution to membrane. Incubate on rocker/shaker for 10 - 20 minutes or until the desired degree of staining has been achieved.
  10. Stop substrate development by pouring off development solution and rinsing membrane in deionized water. Allow membrane to air dry on a paper towel. Blots can be stored dry for long periods of time if protected from light.

Immunostaining Protocol - Rapid Method (2 hours)

  1. After transfer, thoroughly dry the blot by incubating at 37 °C for 30 minutes.
  2. Dilute 1° antibody according to manufacturer's recommendations, in Diluent Buffer.
  3. Add 20 mL of diluted 1° antibody to membrane. Incubate on rocker/shaker at room temperature for 1 hour.
  4. Pour off 1° antibody and wash membrane for 10 seconds with TTBS. Repeat wash.
  5. Dilute 2° antibody 1:8000 - 1:50,000 (or manufacturer's recommended dilution) in Diluent Buffer.
  6. Add 20 mL of diluted 2° antibody to membrane. Incubate on rocker/shaker at room temperature for 30 minutes.
  7. Pour off 2° antibody and wash membrane for 10 seconds with TTBS. Repeat wash.
  8. Dilute alkaline phosphatase (AP) reagent 1:1000 (or manufacturer's recommended dilution) in Diluent Buffer.
  9. Add 20 mL of AP reagent to membrane. Incubate on rocker/shaker at room temperature for 30 minutes.
  10. Pour off AP reagent and wash membrane for 10 seconds with TTBS. Repeat wash.
  11. Prepare Substrate Development Solution by adding 132 µL of NBT stock and 66 µL of BCIP stock to 20 mL of AP Substrate Buffer. Do not allow membrane to dry while preparing Substrate Development Solution.
  12. Add Substrate Development Solution to membrane. Incubate on rocker/shaker for 10 - 20 minutes or until the desired degree of staining has been achieved.
  13. Stop substrate development by pouring off development solution and rinsing membrane in deionized water. Allow membrane to air dry on a paper towel. Blots can be stored dry for long periods of time if protected from light.
Immunostaining Materials
Primary (1°)
Antibody		 Any polyclonal or monoclonal antibody that has been validated for use in western blots
Secondary (2°)
Antibody		 Depends on 1° antibody utilized, for example:
  • Biotinylated rabbit anti-chicken IgG (Jackson ImmunoResearch catalog # 303-065-003, or equivalent)
  • Biotinylated rabbit anti-goat IgG (Jackson ImmunoResearch Cat# 305-065-045, or equivalent)
  • Biotinylated rabbit anti-mouse IgG (Jackson ImmunoResearch Cat# 315-065-045, or equivalent)
  • Biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Cat# 111-065-045, or equivalent)
Streptavidin-alkaline
phosphatase (AP)		 Jackson ImmunoResearch, Cat# 016-050-084, or equivalent
Wash Solution (TTBS) 50 mM Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.4
Blocking Buffer 3% BSA (Fraction V, 3x crystallized), TTBS, 0.2% azide, pH 7.4
Diluent Buffer 1% BSA (Fraction V, 3x crystallized), TTBS, 0.2% azide, pH 7.4
AP Substrate Buffer 0.1 M Tris, 0.1 M NaCl, 5 mM MgCl2, pH 9.5
NBT Stock 50 mg/mL NBT in 70% DMF
BCIP Stock 50 mg/mL BCIP in 100% DMF
Additional Tools Forceps, clean plastic test tube, plastic trays, gloves, pipettes

Hints

  • Always use forceps and wear gloves when handling gels, membranes and filter paper
  • Minimize trapped air bubbles by laying gels, membranes and filter paper on transfer stack at an oblique angle.
  • Use the appropriate percentage SDS-PAGE gel for protein of interest
  • Gel, membrane and filter paper should all have same dimension. If membrane is smaller than gel, part of gel may not be transferred. If membrane or filter papers are larger than gel it may result in uneven current distribution and consequently uneven protein transfer.
  • Use pre-stained molecular weight markers to check the transfer efficiency.
  • Check non-specific binding by running extra sample lane and omitting 1° antibody from development procedure. Or run with a non-specific antibody control in place of 1° antibody.