Investigating S-acylation of SARS-Cov-2 Spike Protein Leads to New Insights into Viral Infectivity

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COVID-19 Fluorescence imaging Imaging Western Blotting

A better understanding of coronavirus biology can enable development of new antivirals to help stop COVID-19 and prevent future pandemics. In recent work, Puthenveetil et al. characterized S-acylation of the Spike (S) protein of SARS-CoV-2. This post-translational modification is known to be important to the viral replication cycle of other viruses across multiple virus families but has not been studied in SARS-CoV-2.

To control for gel loading and S protein expression in the S-acylation experiments, the authors used the Azure Sapphire Biomolecular Imager to detect GAPDH by NIR fluorescence and SARS-CoV-2 S protein by chemiluminescence on Western blots (Figure 1).

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.

The authors expressed S protein in cultured cells and carried out metabolic labeling with a fatty acid that was detected using a rhodamine-labeled fluorescent probe. They found substantial S-acylation that was blocked by 2BP, a global inhibitor of S-acylation. The authors also found the S-acylation of the S protein was dependent on the presence of the cysteines in the C-terminal domain.

Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay
Figure 1 from Puthenveetil R, Lun CM, Murphy RE, et al. (2021) S-acylation of SARS-CoV-2 Spike protein: mechanistic dissection, in vitro reconstitution and role in viral infectivity. Licensed under CC BY 4.0. The Sapphire was used to image chemiluminescent and NIR Western blots to control for protein loading and S-protein expression in the S-acylation assay (panel B).

Further experiments expressing mutated versions of the S protein identified which cysteines were S-acylated, the effect of mutating these cysteines on particle infectivity, and which members of the human family of enzymes that carry out S-acylation were able to modify the S protein in cells and in an in vitro assay.

What is S-acylation?

S-acylation involves adding long-chain fatty acids to cysteine residues on the cytosolic side of transmembrane proteins. The cytoplasmic tail of the SARS-CoV-2 S protein contains 10 cysteines in 6 potential S-acylation sites. Puthenveetil et al note that all but one of these are conserved with SARS-CoV, and most are conserved with other coronaviruses that infect humans, including MERS. Still, nothing is known about what role, if any, the S-acylation of these cysteines may play in the biology of viruses, such as SARS-COV-2.

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In addition to chemiluminescence and near-infrared fluorescence imaging, the Sapphire provides densitometry, phosphor, multichannel fluorescence, 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.

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.