Western Blotting Reagents Roundup – September 2020

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The Reagent Roundup is made of brief summaries of publications in which researchers used Azure Biosystems reagents for Western blotting and Western blot quantitation in their studies. It is published every quarter. This quarter’s Reagent Roundup features publications from Duke University School of Medicine, University of Minho School of Medicine, the National Institutes of Health, and Boys Town National Research Hospital.

Adipocyte REVERBα dictates adipose tissue expansion during obesity

Chemiluminescent Western blot Azure Biosystems Protein-Free Blot Blocking Buffer showing REVERBα expression in adipose tissue over 24 hours
Figure F. Western blot blocked with Azure Biosystems Protein-Free Blot Blocking Buffer showing REVERBα expression in adipose tissue over 24 hours.

In a recent pre-print, Hunter et al recently demonstrated that REVERBa, a circadian clock component, modulates white adipose tissue metabolism in response to changes in metabolic state. The researchers created a mouse model in which REVERBa was knocked out in only white adipose tissue and compared these mice to mice missing REVERBa in all tissues. Chemiluminescent and fluorescent Western blots were used to confirm protein changes in knock out mice, and to follow circadian changes in protein levels. All Western blot experiments used Azure’s Protein-Free Blocking Buffer to block nitrocellulose membranes. This blocking buffer is a great choice to enhance signal and reduce background for general Western blots and is compatible with all detection chemistries.

SHOP: Protein-free blocking buffer

Molecular Targeting of Cancer-Associated PCNA Interactions in Pancreatic Ductal Adenocarcinoma using a Cell-Penetrating Peptide

Figure 4. Pancreatic cancer cells were treated with 50 μM R9-caPep for up to 48 h. Representative Western blot analysis was performed to analyze both phosphorylated γ-H2AX and unphosphorylated H2AX. Actin was used as the loading control and were detected by ECL prime or AzureSpectra 800 secondary antibodies and imaged on the Azure c600.
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Smith et al identified a novel potential therapeutic approach for pancreatic cancer. In Molecular Therapy Oncolytics, the researchers examined whether a peptide that mimics a part of the proliferating cell nuclear antigen (PCNA) could interfere with PCNA-protein interactions in pancreatic cancer cells. They found that the peptide killed cancer cells by inhibiting DNA replication and DNA repair, resulting in the accumulation of DNA damage. Fluorescent Western blots were used to assess DNA damage by detecting phosphorylated histone H2AX, a biomarker for double stranded DNA breaks. The secondary antibody used was the Azure Spectra 800 Secondary Fluorescent Antibody.

SHOP: Azure Spectra 800 Secondary Fluorescent Antibody.

Poxvirus-encoded decapping enzymes promote selective translation of viral mRNAs

Cantu et al identified a surprising new function of vaccinia virus decapping enzymes. The decapping enzymes D9 and D10 are known to promote degradation of viral and cellular mRNAs. In PLOS Pathogens, the researchers compared genome-wide translation efficiency in cells infected with wild type vaccinia virus vs mutant virus lacking functional decapping enzymes. The results indicated that paradoxically, while promoting degradation of mRNAs, the decapping enzymes are also required for selective translation of certain post-replicative viral proteins whose mRNAs have 5’-poly(A) leaders. Translated proteins were detected on chemiluminescent western blots using Azure HRP-conjugated Secondary Antibodies.

SHOP: Azure HRP-conjugated Secondary Antibodies

2‐Oxothiazolidine‐4‐carboxylic acid inhibits vascular calcification via induction of glutathione synthesis

Patel et al are investigating the potential of a cysteine prodrug, 2‐oxothiazolidine‐4‐carboxylic acid (OTC) to inhibit arterial medial calcification. In a recent article published in the Journal of Cell Physiology, the researchers used Azure’s Radiance ECL HRP substrate to detect markers of cell differentiation in cultured vascular smooth muscle cells (VSMC), grown in a calcifying medium with or without OTC. Western blotting revealed that treatment with OTC reversed the increase in osteoblast markers and rescued the drop in glutathione synthesis enzymes observed in calcifying cells, leading the authors to conclude the drug was effective in vitro and might have clinical relevance. The long-lasting signal and high sensitivity of Radiance ECL substrate allows for reliable comparisons of protein levels between samples.

Free sample: Radiance ECL substrate

You can find more publications using Azure reagents and imaging systems on the Azure publications list, or contact Azure for assistance identifying publications using a specific product.

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