How the Sapphire Scanner is Used to Better Grocery Store Tomatoes

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Imaging Publication Spotlight 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 from the Institute of Olive Tree in Greece 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.

Growing the tomatoes used in this study

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.

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.

The role of temperature in mediating postharvest polyamine homeostasis in tomato fruit
Chemiluminescent Western blot showing the role of temperature in mediating postharvest polyamine homeostasis in tomato fruit

How the research was conducted

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.

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

Sapphire FL biomolecular imager
The new Sapphire FL images phosphor screens with high sensitivity. With customizable and user-changeable laser and filter modules, it easily adapts to a lab’s changing needs and advancing research.

Learn more about the Sapphire imager and how it can support your research by requesting a quote.

The findings

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.

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

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Fluorescence imaging Troubleshooting Western Blotting

Keep reading if you’re having problems using ponceau! 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. You can use protein stains to visualize all of the protein bands on a gel or membrane. Staining the blot with some total protein stain can provide a standard for total protein normalization of quantitative Western blots.1

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

  • Azure Quick Tip: To avoid high background due to Ponceau staining, consider using other total protein stains.

    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

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.

If you’re set on using Ponceau, choose Azure Ponceau to detect total protein transferred to membranes. This stain is reversible and can be used to check the efficiency of protein transfer before immunodetection. Each 500 ml contains enough stain for 50 standard blots that are around 9×7 cm.

Shop Ponceau Stain Alternatives, like Azure Ponceau Stain

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.