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Methods:Western Blotting

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Contents

Purpose

To identify the presence/amount of a specific protein using an immunodetection

Materials Needed

Equipment

  • SDS gel
  • Whatman paper (filter paper)
  • nitrocellulose paper
  • Electro- transfer apparatus

Solutions

Electroblot Buffer
Dissolve 9.09 g Tris, 43.2 g Glycine, and 600 ml Methanol into 2.4 L deionized H2O. Do not add acid or base to adjust pH of the buffer.
Tris-Buffered Saline (TBS)
20 mM Tris-HCl, 500 mM NaCl pH 7.5 (2 L total volume)
Dissolve 4.84g Tris, 58.48g NaCl into 1.5 L deionized H2O, pH to 7.5 with HCl
Wash Solution (TTBS)
20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20, pH 7.5
Add 0.5 ml Tween 20 to 1 L of TBS
Blocking Solution (1% BSA-TBS or 1% dry milk-TBS)
1% solution of BSA or Carnation non-fat dry milk in TBS
Add 1.0 g BSA or dry milk to 100 ml TBS. Stir to dissolve.
Antibody Buffer (1% BSA-TTBS)
Add 2 g BSA to 200 ml TTBS. Stir to dissolved.
Color Development Buffer
0.1 M Tris, 0.5 mM MgCl2, pH 9.5 (1 L total volume)
Dissolve 0.102 g MgCl2*H2O and 12.1 g Tris into 800 ml deionized H2O, Adjust pH to 9.5 with HCl.
NBT solution
Prepare 1.0 ml 70% DMF (0.7 ml dimethylformamide with 0.3 ml deionized H2O). Dissolve 30 mg of NBT (nitroblue tetrazolium) into 70% DMF.
BCIP solution
Dissolve 15 mg BCIP (5-Bromo-4-chloroindolyl phosphate) in 1.0 ml DMF
Color Development Solution
Mix 1 ml NBT solution and 1 ml BCIP solution with 100 ml Color Development Buffer. Use immediately.

Protocol

Kits have become popular for doing this. The very vague, general idea is written below:

  1. run an SDS gel of the sample
  2. transfer the contents of the gel over to a nitrocellulose or poly(vinylidene difluoride) membrane
  3. wash the blot with blocking solution-This is to prevent the antibody from directly binding to the membrane so avoid touching the blot with your hands as well.
  4. probe with the 1˚ antibody
  5. wash blot to remove excess 1˚
  6. probe with a conjugated 2˚ antibody (normally conjugated to Horseradish Peroxidase or Alkaline Phosphatase/ CL)
  7. wash blot to remove excess 2˚
  8. add substrate for conjugated protein and develop the blot

Links

User notes

References

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