Problems with Ponceau? Consider Alternative Total Protein Stains for Fluorescent Western Blots

Troubleshooting Fluorescence imaging Quantification Western Blotting
Ponceau S is a fast, reversible protein stain often used to confirm that protein samples have successfully transferred from the gel to the membrane before a researcher moves ahead with the time-consuming process of immunoblotting.

Staining the membrane after transfer makes it possible to quickly and easily identify any problems such as incomplete or uneven transfers, or artifacts due to the presence of air bubbles, before probing the blot with antibodies. In addition, staining the blot with some total protein stains can provide a standard for total protein normalization of quantitative Western blots.1

However, though Ponceau staining is reversible, it is not compatible with fluorescent Western blot detection. Even after thorough destaining, Ponceau stain can leave an autofluorescent residue on the membrane that increases background fluorescence.

The figure below shows a multicolor fluorescent Western blot. The right half of the blot was stained using Ponceau and then destained before immunoblotting.

High background when fluorescent western blot stained with ponceau

Both halves of the blot were then blocked with Azure Fluorescent Blot Blocking Buffer and probed for four proteins using four different fluorescent probes as labeled in the figure. The blot was imaged using the Azure Imager RGB module which assesses Cy2, Cy3, and Cy5-compatible channels. Despite thorough destaining, a very high fluorescent High background when NIR western blot stained with ponceaubackground is seen on the half of the blot that was stained with Ponceau.

The figure to the right shows the same blot imaged using the Azure cSeries NIR module, which assesses 700nm and 800nm channels. The fluorescent background is reduced in the NIR-imaged blot, but is still substantial compared to the unstained half of the blot.

To avoid high background due to Ponceau staining, consider using other total protein stains.2 AzureRed Fluorescent Total Protein Stain is completely compatible with downstream Western blotting detection, including fluorescent detection, and with downstream mass spectrometry.


Learn more about AzureRed Fluorescent Total Protein Stain
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  1. Thacker JS, et al. Total protein or high-abundance protein: Which offers the best loading control for Western blotting? Biochem. 2016; 496:76-78. PMID: 26706797.
  2. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. 2017;17:1600189. PMID: 28941183.