Western Blotting

For analysis, proteins are usually separated by polyacrylamide gel electrophoresis. Another characterization method is the subsequent Western Blot followed by immuno detection. For this, proteins are blotted onto a membrane by electro transfer. The transfer can be done either by semi-dry or tank blotting.

Blotting 305 x 120

Semi-dry Blotting

  • Usually performed with a discontinuous buffer system
  • For efficient and fast protein transfer (within 15 min): SERVA Xpress blotting with a special buffer system and blotting fleeces
  • Allows even transfer of large and small proteins

Tank Blotting

  • Usually performed with Towbin buffer
  • Small proteins are transferred faster than big ones

Both, nitrocellulose (NC) and PVDF membranes can be used as transfer membranes.

Nitrocellulose (NC) – Often used for routine applications

  • Binding capacity: 80 - 120 mg/cm2
  • Low background
  • „Fragile“ if not supported
  • Protein binding depends on the methanol concentration
  • For proteins, DNA, RNA

PVDF – Superior transfer results compared to NC due to higher binding capacity

  • Binding capacity: 125 - 250 mg/cm2
  • Hydrophobic
  • Mechanically stable
  • For proteins

Detection of membrane-bound proteins is usually carried out with horseradish peroxidase (HRP) or alkaline phosphatase (AP) -labelled antibodies.
Then, antigen-bound antibodies are detected by a chemical reaction of the enzyme with the respective substrate which produces a chemiluminescent or colorimetric signal.

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