How the Sapphire Scanner is Used to Better Grocery Store Tomatoes

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Imaging Western Blotting

Few things are more disappointing than a tasteless, mealy grocery store tomato. These bland fruits are pale imitations of the vine-ripened tomatoes available from the garden at the end of summer. Tomatoes are perishable, and providing ripe, high-quality tomatoes that maintain their texture and nutritional content is a challenge for commercial growers.

recent publication by Tsafouros et al provides a window into the intense, ongoing research aimed at understanding how and why tomatoes and other fruits ripen under various conditions. Such studies could improve the postharvest shelf-life and the quality of commercially grown tomatoes.

The authors carried out an exhaustive biochemical and molecular biological characterization of polyamine metabolism in the tomatoes. They assessed the total content of a variety of polyamines, the activity of the enzyme responsible for breaking down polyamines, the expression of all 23 genes encoding factors known to be involved in polyamine metabolism, the levels of the proteins involved in polyamine synthesis, and the levels of hydrogen peroxide, a biproduct of amine oxidases acting on polyamines. Protein levels were measured by chemiluminescent western blots imaged using the Sapphire Biomolecular Imager from Azure Biosystems.

Tomatoes are typically picked before ripening and stored and transported at low temperatures in an attempt to increase the shelf-life. Tsafouros et al examined the effect on ripening of storing picked tomatoes at various temperatures for a week. The authors also characterized in detail the effect of storage temperature on the metabolism of polyamines, compounds known to play a role in fruit ripening and the content of which are known to be associated with tomato quality and shelf life.

For this study, tomatoes were grown in a greenhouse and either picked at “commercial maturity” (when the tomato is just turning color) or left on the plant to mature for an additional week. The harvested tomatoes were stored at 5, 10, or 25 °C and after 7 days were compared to each other and to tomatoes left to ripen on the vine.

Their results demonstrate that cold storage alters polyamine metabolism, and support storage of tomatoes at 10 °C after picking at commercial maturity. Lower temperatures appeared to induce a stress response, perhaps to protect against chilling injuries, while higher temperatures were associated with lower polyamine levels and lower quality fruit.

The Sapphire imager provides multiple imaging capabilities including multi-channel fluorescence, white light, phosphor imaging, densitometry and chemiluminescence imaging of blots, gels, tissues, microplates, and more.

Learn more about the Sapphire imager and how it can support your research by clicking here.

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

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Fluorescence imaging Quantification Troubleshooting 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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SOURCES

  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.