Protein analysis

Dot blots

Dot blotting is a simple, convenient method for detection of proteins in crude lysates or solutions without the need for separation by SDS-PAGE. This method is especially useful as a simple control because it avoids problems that may be due to the western transfer process. Any components that interfere with binding or bind nonspecifically, however, will not be spatially separated from the protein and will interfere with the intensity of signals. Suitable controls should always be employed to compensate for this.

Materials required

  • Nitrocellulose membrane (e.g., Schleicher and Schuell BA85)
  • Protein samples
  • Dilution buffer for native or denaturing conditions (see table Dilution buffer for denaturing conditions or Dilution buffer for native conditions)
Dilution buffer for denaturing conditions
Composition of working solution, pH 8.0 Component Amount per liter
8 M urea Urea 480.5 g
100 mM NaH2 PO4 NaH2 PO4·H2O 13.8 g
10 mM Tris·Cl Tris base 1.2 g

Dilution buffer for native conditions
Composition of working solution, pH 8.0 Component  Amount per liter
50 mM NaH2PO4 NaH2PO4·H2O 17.5 g
300 mM NaCl  NaCl 6.9 g

  1. Dilute protein samples in buffer to final protein concentrations of 1–100 ng/µl.

    Tip: The protein of interest is diluted in dilution buffer for denaturing conditions, dilution buffer for native conditions, or another preferred buffer.

  2. Apply 1 µl samples of diluted protein directly onto membrane. It is also possible to use crude cell lysate and apply 1 µl samples with an estimated concentration of 1–100 ng/µl protein.

    Note: Under native conditions especially, the antibody epitope must be at least partially exposed to allow antibody binding. In most cases diluting the protein with buffer containing denaturing reagents will increase epitope exposure and give better results.

    Tip: To differentiate between nonspecific and positive signals, an extra sample containing 1 µl of a cell extract of the host strain without plasmid (or other suitable control) should also be applied to the membrane and treated together with the protein of interest.

  3. After applying the samples, the membrane should be dried for a short time at room temperature before proceeding with the detection process.

    Tip: For larger sample volumes, suitable equipment is available from several suppliers.

  4. Proceed with immunodetection (see Immunodetection using a chemiluminescent detection method or Immunodetection using a chromogenic detection method).
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