Potential Treatment for Advanced Kidney Cancer

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Protein Assays Western Blotting

Renal cell carcinoma (RCC), the most common type of kidney cancer, is aggressive and frequently develops resistance to therapy. Advanced RCC has a poor prognosis and new, effective treatment strategies are badly needed. Metastatic RCC may be treated with sunitinib, but the majority of cancers eventually develop resistance to this drug. Sunitinib inhibits signaling through receptor tyrosine kinases, interfering with pro-growth signals received by the tumor.

In a recent publication, Markowitsch et al investigated the effect of shikonin (SHI) on sunitinib-sensitive and sunitinib-resistant RCC cell lines in cell culture. SHI is a naturally occurring compound. It’s an active component of a dried plant root (Lithospermum erythrorhizon) that has been used in traditional Chinese medicine to address a variety of ailments.

Earlier studies have demonstrated the anti-cancer capabilities of SHI and have shown that SHI can enhance the activity of traditional chemotherapeutics or re-sensitize chemotherapy-resistant cells to therapy. How SHI exerts these effects is not clear; however, as SHI has been found to affect many cell signaling pathways and induce cell death via apoptosis and necroptosis.

The recent work by Markowitsch et al thoroughly examined the effect of SHI on many aspects of RCC cell biology. Several assays relied on imaging with the Azure Sapphire Biomolecular Imager, including studies that characterized protein expression related to multiple signaling pathways by Western blot, adhesion of RCC cells to extracellular matrix proteins and to vascular endothelial cells, and studies of tumor cell migration and chemotaxis, relied on imaging with the Sapphire.

Figure 2 from Markowitsch et al. (2022) Shikonin inhibits cell growth of sunitinib-resistant renal cell carcinoma by activating the necrosome complex and inhibiting the AKT/mTOR signaling pathway. Licensed under CC BY 4.0. The Azure Biomolecular Imager was used to image and quantify RCC colonies on cell culture dishes.

The authors took advantage of several imaging modes provided by the Sapphire. The Sapphire was used to image and quantify the growth of colonies in 6-well culture dishes using Coomassie Blue dye detection. The Sapphire was used to assess cell adhesion, chemotaxis and cell motility by measuring the fluorescence of cells labeled with CellTracker Green in either pre-treated 24-well culture dishes or on the lower surface of membrane inserts in 24-well plates. Western blots were detected using enhanced chemiluminescence and imaged on the Sapphire.

The results of the numerous studies indicated that, though the specific effects varied by cell line, SHI had antitumor effects on all cell lines studied. SHI was found to inhibit RCC cell growth, proliferation, and clone formation, both in sunitinib-sensitive and sunitinib-resistant cell lines. SHI caused cell cycle arrest and induced cell death, primarily via necroptosis. SHI also inhibited the AKT/mTOR pathway, which presents another mechanism by which SHI may interfere with RCC cell survival and growth.

The authors conclude the data is promising and that SHI should be further studied as a potential addition to therapy for patients with advanced and therapy-resistant RCC.

In addition to visible and fluorescent imaging of tissue culture plates, and chemiluminescent imaging of Western blots, the Sapphire Biomolecular Imager provides densitometry, phosphor, multichannel fluorescence, near-infrared, and white light imaging of blots, gels, tissues, and more. Learn more about the Sapphire Imager and how Azure can support your research by clicking here.

 

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What is Western Blotting?

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Fluorescence imaging Quantification Troubleshooting Western Blotting

Your common Western blotting questions, answered.

Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex mixture of proteins. It is a powerful tool that provides information about the presence, size, and under the right conditions, even the amount of a protein. Though commonly used and often routine in many labs, Western blotting can be source of frustration when it doesn’t work. It involves several steps, each of which needs to be optimized to achieve the best results. The key to the best Westerns is understanding the process. We’re here to help with some answers to your most commonly asked Western blotting questions.

What are some of the detection methods used in Western blotting?

Several options are available to detect Western blots. Chemiluminescence is likely the most common. Other means of detection include fluorescence, near-infrared fluorescence, colorimetric, and radioactive.

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What is chemiluminescent detection?

In Western blotting, chemiluminescent detection is a method of detecting the location of antibodies bound to a Western blot. Chemiluminescent detection relies on an enzyme, either horseradish peroxidase or alkaline phosphatase, bound to an antibody. The enzyme converts a substrate to a product that emits light (chemiluminescence). The light emitted can be detected on X-ray film or by CCD camera.

What's more sensitive: chemiluminescence or fluorescence?

In general, fluorescent detection can detect picograms of protein while chemiluminescence can detect protein in the femtogram range.

However, sensitivity of detection depends on many things. The ability to detect small amounts of target protein requires a high-quality primary antibody with high affinity and specificity for the target protein. In addition, with CCD cameras, very long exposures are possible to maximize the chance of detecting a low-abundance band but this requires minimizing background “noise” on the Western blot. In addition, different fluorophores have different quantum yields, and some HRP substrates are engineered to increase sensitivity, so the sensitivity of fluorescent detection depends on the specific fluorophore used, and the sensitivity of chemiluminescent detection depends on the substrate used.

Radiance Q chemiluminescent substrate is designed to produce a strong, long-lasting signal for large linear dynamic range and quantitative data

Continue readingBeginning Chemiluminescent Western Blotting
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What are the advantages of using fluorescent Western blot vs. chemiluminescent Western blot?

Multiplex detection is possible by using two or more fluorescent dyes on an instrument that can excite and detect light from each dye
Multiplex detection is possible by using two or more fluorescent dyes and an instrument that can excite and detect the light from each dye.

1. Fluorescent Western blotting allows multiplexing. By using different fluorescent dyes with non-overlapping excitation and emission spectra, multiple proteins can be assayed on one blot without needing to strip and re-probe the blot.

2. Fluorescent detection is more quantitative than chemiluminescent detection. Chemiluminescent detection relies on an enzyme (HRP or AP) bound to the antibody, and the activity of the enzyme can change depending on conditions and as the amount of substrate changes. Fluorescent detection relies on the emission of light from a fluorescent probe bound to the antibody. The fluorescence intensity will only depend on the number of fluorescent molecules present in a given spot.

Continue readingAlternative Total Protein Stains for Fluorescent Western Blots

Is HRP a chemiluminescent substrate?

The principle of chemiluminescent Western blotting
The principle of chemiluminescent Western blotting

No! Even though it is an important component of chemiluminescent detection, HRP stands for horseradish peroxidase, an enzyme isolated from the roots of the horseradish plant. HRP catalyzes the oxidation of substrates, transferring electrons from the substrate to peroxide. In chemiluminescent Western blot detection, HRP is conjugated to an antibody and the location of the antibody on a blot is detected by incubating the blot with a substrate that will produce light after it is oxidized by the HRP enzyme.

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What is a chemiluminescence substrate?

luminol chemical formula
Luminol chemical formula

Chemiluminescent substrates produce light in the presence of HRP and hydrogen peroxide. An example of a chemiluminescent substrate is luminol. Luminol is oxidized to 3-aminophthalate which emits light (chemiluminescence) that can be detected on X-ray film or by a CCD camera.

Additional Western Blotting Resources

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N-terminomics of SARS-CoV-2

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COVID-19 Imaging

A better understanding of the interactions between SARS-CoV-2 and the host cells it infects could lead to new approaches to treat or prevent infections. Proteolysis is essential to the SARS-CoV-2 life cycle; two viral polyproteins must be cleaved to generate components of the viral replication/transcription complex. The polyproteins are snipped apart by two viral proteases, the papain-like protease (PLP) and main protease (Mpro).

PLP and Mpro also cleave cellular proteins, which may help the virus by modifying the activities of these targets. Protease inhibitors have demonstrated antiviral activity in cell culture and are a subject of anti-coronavirus drug development. To this end, understanding the complete set of proteolytic changes that occur upon viral infection would be important.

Meyer et al carried out an unbiased study of proteolysis of viral and cellular proteins during viral infection in cell culture. Proteins were extracted from infected and uninfected cells, differentially labeled, and analyzed by mass spectrometry in an N-terminomics approach that identified neo-N-termini. In doing so, several cellular targets of viral proteases were identified as well as new viral protein cleavage sites, which hold potential for future vaccine or antibody development.”

Figure 4g from Meyer et al. (2021) Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential. Licensed under CC BY 4.0. The Azure c600 was used to capture images of a chemiluminescent Western blot, demonstrating the cleavage of cellular proteins PAICS and GOLGA3 in transfected cells expressing SARS-CoV-2 Mpro.

To confirm their findings, both in vitro and cell-based assays were used to observe the process of SARS-CoV-2 proteases cleaving cellular proteins. Specifically, SARS-CoV-2 Mpro was expressed in cells with the subsequent cleavage of two target proteins being confirmed via chemiluminescent Western blotting imaged on an Azure c600.

The authors found that the cleavage of cellular proteins was important for viral infectivity. To test for this, RNAi was used to deplete the levels of 14 potential viral protease substrates. Surprisingly, reducing each of the 14 proteins resulted in reduced viral titers, suggesting that viral proteases target and cleave cellular proteins to increase pro-viral activities. Consistent with this, two inhibitors of cellular targets were found to potently inhibit infection of cells by SARS-CoV-2, indicating the activity of these cellular proteins is important to viral infectivity.

This study demonstrates the enormous potential of characterizing proteolysis in the context of viral infection to aid in the development of targeted anti-viral strategies.

In addition to chemiluminescence imaging, the Azure 600 imager provides multicolor fluorescence, white light, and two-channel near-infrared imaging of blots, gels, and more.