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Prepare Membrane

Prepare Membrane for Protein Identification

For Staining and Immunodetection

PVDF membrane should be washed with deionized water to remove any gel debris. The blot can then be incubated with the blocking solution.

For Rapid Immunodetection and Visualization by Transillumination

After the transfer is complete, PVDF membranes can be dried before continuing on to staining or immunodetection procedures. Drying enhances the adsorption of the proteins to the PVDF polymer, helping to minimize desorption during subsequent analyses. As the blotted membrane dries, it becomes opaque. This optical change is a surface phenomenon that can mask retention of water within the depth of the pores. The membrane should be dried for the recommended period to ensure that all liquid has evaporated from within the membrane’s pore structure (refer to the membrane Drying Methods Protocol, later in this section).

Storage

Membranes can be stored dry for long periods of time after proteins have been transferred with no ill effects to the membrane or the proteins (up to two weeks at 4°C; up to two months at -20°C; for longer periods at -70°C). Some proteins, however, may be sensitive to chemical changes (e.g. oxidation, deamidation, hydrolysis) upon prolonged storage in uncontrolled environments. Long term storage at low temperature is recommended. Prior to further analysis, dried membrane must be wet by soaking in 100% methanol for PVDF membranes or Milli-Q water for nitrocellulose membranes.

Protein Visualization

Transillumination

Transillumination (see “Calf Liver Protein Stains” figure on the following page) is a visualization technique unique to PVDF membranes and was first described for Immobilon-P transfer membrane (Reig and Klein, 1988). This technique takes advantage of a characteristic unique to PVDF membranes; areas of PVDF coated with transferred protein are capable of wetting out in 20% methanol while the surrounding areas of PVDF are not. In the areas where the PVDF wets, it becomes optically transparent, allowing visualization of protein bands using backlighting and photographic archiving. The process is fully reversible by evaporation. Further denaturation of the proteins is unlikely as the proteins had been previously exposed to methanol during blotting. Even though this technique does not allow for visualization of low abundance proteins, it can be used to assess the overall transfer efficiency and the suitability of the blot for further analysis.

Staining

Staining (see “Calf Liver Protein Stains” figure below) is a simple technique to make proteins visible on a blot. Staining can be used to:

  • Verify that proteins have transferred onto the membrane
  • Determine if the lanes were loaded equally
  • Evaluate the overall efficiency of the transfer, especially for a new buffer system or protein
  • Identify and excise bands for peptide sequencing
Many types of stains are available including organic dyes (Ponceau-S red, amido black, fast green and Coomassie Blue), fluorescent dyes (fluorescamine, courmarin) and colloidal particles (gold, silver, copper, iron and India ink) (Kurien et al., 2003). The table below lists the most common stains for detection of total proteins on western blots. The dyes are separated into two groups: reversible and irreversible stains.

Reversible Stains

Reversible stains allow assessment of the blot and then can be washed from the membrane. These will not interfere with subsequent immunodetection or other analysis of the proteins on the blot. The most commonly used reversible protein stain is Ponceau-S red. The major drawback of reversible stains is that they are less sensitive than irreversible stains. Since the staining pattern of more abundant proteins in a blot is generally a good indicator of how well low abundance proteins transferred, this drawback can be minimized in most cases.

Fluorescent blot stains are highly sensitive and compatible with downstream immunodetection, Edmanbased sequencing and mass spectrometry (Berggren et al., 1999). Sypro Ruby and Sypro Rose protein blot stains (Invitrogen) can be used prior to chromogenic, fluorogenic or chemiluminescent immunostaining procedures and provide sensitivity of about 1-2 ng/band (Haugland, 2002).

Irreversible Stains

Irreversible stains generally exhibit the best sensitivity but can interfere with or prevent further analysis of the proteins. Examples of irreversible stains are amido black and Coomassie Brilliant Blue.

Calf liver proteins were visualized after electroblotting to Immobilon-P membranes: (A) Transillumination, (B) Coomassie™ Brilliant Blue, (C) Ponceau-S red, (D) Amido black and (E) CPTS total protein stains. Left to right, molecular weight standards and 12.2 μg, 6.1 μg, 3.1 μg of the lysate per lane.

Common Stains Used in Western Blotting and Their Attributes
Detection Reagent Approximate Sensitivity (protein per spot) Reference
Reversible Ponceau-S 5pμ Dunn et al., 1999
Fast Green FC 5pμ Dunn et al., 1999
CPTS 1pμ Bickar et al., 1992
Sypro Ruby 1-2 ng Haugland, 2002
Sypro Rose 1-2 ng Haugland, 2002
Irreversible Amino black 10B 1μg Dunn et al., 1999
Coomassie Brilliant Blue 250 500ng Dunn et al., 1999
India Ink 100ng Dunn et al., 1999
Colloidal gold 4ng Dunn et al., 1999

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