Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

Abstract Objective CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity. Results We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPβ isoforms.

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Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

10.21203/rs.2.11944/v2 ◽

2019 ◽

Author(s):

Hamood AlSudais ◽

Nadine Wiper-Bergeron

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Transcription Factors ◽

Western Blot ◽

Light Chain ◽

Myosin Light Chain ◽

Intense Band ◽

Cellular Processes ◽

Enhancer Binding Protein ◽

Myoblast Cells

Abstract Objective CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity. Results We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of the C/EBPβ LIP isoform.

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Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

10.21203/rs.2.11944/v1 ◽

2019 ◽

Author(s):

Hamood AlSudais ◽

Nadine Wiper-Bergeron

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Transcription Factors ◽

Western Blot ◽

Light Chain ◽

Myosin Light Chain ◽

Intense Band ◽

Cellular Processes ◽

Enhancer Binding Protein ◽

Myoblast Cells

Abstract Objective CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity. Results We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of the C/EBPβ LIP isoform.

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Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

10.1101/2020.07.21.213488 ◽

2020 ◽

Author(s):

Manish Bhattacharjee ◽

Navin Adhikari ◽

Renu Sudhakar ◽

Zeba Rizvi ◽

Divya Das ◽

...

Keyword(s):

Mass Spectrometry ◽

Plasmodium Falciparum ◽

Western Blot ◽

Wild Type ◽

Malaria Parasites ◽

Post Translational Modifications ◽

Functional Conservation ◽

Cellular Processes ◽

Physiological Substrates

ABSTRACTA variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (Δrub1) or NEDD8 conjugating E2 enzyme (ΔUbc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

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Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

Biochemistry ◽

10.1021/bi00392a046 ◽

1987 ◽

Vol 26 (18) ◽

pp. 5885-5890 ◽

Cited By ~ 7

Author(s):

Mary H. Nunnally ◽

Donald K. Blumenthal ◽

Edwin G. Krebs ◽

James T. Stull

Keyword(s):

Skeletal Muscle ◽

Monoclonal Antibody ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Binding Domain ◽

Rabbit Skeletal Muscle ◽

Skeletal Muscle Myosin ◽

Calmodulin Binding ◽

Muscle Myosin

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Identification of N-terminal modification for recombinant monoclonal antibody light chain using partial reduction and quadrupole time-of-flight mass spectrometry

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.2790 ◽

2006 ◽

Vol 20 (24) ◽

pp. 3674-3680 ◽

Cited By ~ 17

Author(s):

Lei Yu ◽

Richard L. Remmele ◽

Bing He

Keyword(s):

Mass Spectrometry ◽

Monoclonal Antibody ◽

Light Chain ◽

Time Of Flight ◽

Partial Reduction ◽

Recombinant Monoclonal Antibody ◽

Flight Mass Spectrometry

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SIRT1-mediated deacetylation of NF-κB inhibits the MLCK/MLC2 pathway and the expression of ET-1 thus alleviating the development of coronary artery spasm

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00366.2020 ◽

2020 ◽

Author(s):

Bo-Wen Wu ◽

Mi-Shan Wu ◽

Yu Liu ◽

Meng Lu ◽

Jin-Dong Guo ◽

...

Keyword(s):

Coronary Artery ◽

Western Blot ◽

Light Chain ◽

Blot Analysis ◽

Western Blot Analysis ◽

Myosin Light Chain ◽

Coronary Artery Spasm ◽

Luciferase Reporter ◽

Sirt1 Expression

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that cause total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by RT-qPCR and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation VSMCs, and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, EdU assay, RT-qPCR and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and up-regulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.

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Liquid Chromatography-Tandem Mass Spectrometry for Detecting Myosin Light Chain 3 in Dry-Aged Beef

Separations ◽

10.3390/separations8110219 ◽

2021 ◽

Vol 8 (11) ◽

pp. 219

Author(s):

Heeyoung Lee ◽

Yoonji Heo ◽

Jong-Chan Kim ◽

You-Shin Shim

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

Western Blotting ◽

Light Chain ◽

Myosin Light Chain ◽

Muscle Protein ◽

Tandem Mass ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a more accurate technique for detecting proteins than electrophoresis-based methods such as western blotting. Because of its convenience, western blotting is commonly used for protein analysis in beef. We developed a method for detecting myosin light chain 3 (myl3) in beef samples, particularly dry-aged beef, using LC/MS/MS for quality testing. Musculus longissimus dorsi of Holstein was aged for 0, 2, 4, 5, 9, 11, 17, 20, and 24 weeks and used to measure the myl3 concentration. Because of the high molecular weight of myl3, the limitations of LC/MS/MS were overcome by implementing immunoprecipitation and digestion steps. Ultimately, a tryptic fragment of myl3 (13-mer), generated using immunoprecipitation and digestion by a biotinylated antibody, was detected using LC-MS/MS in positive ion mode through multiple reaction monitoring and analyte separation on a C18 column. Our method showed limits of detection and quantification of less than 0.3 and 0.8 μg/kg, respectively. However, differences in the myl3 concentrations according to the aging time were not significant (p > 0.05). After 12 weeks, myl3 disappeared in tested all samples, thus our analytical method can be used for accurate measurement of muscle protein in beef samples.

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Integrating bioinformatics tools to investigate protein phosphorylation

10.7287/peerj.preprints.916 ◽

2015 ◽

Author(s):

Dimitrios Vlachakis ◽

Elena Bencurova ◽

Mangesh Bhide ◽

Sophia Kossida

Keyword(s):

Mass Spectrometry ◽

Western Blot ◽

Protein Phosphorylation ◽

Gel Electrophoresis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Phosphorylation Sites ◽

Post Translational Modifications ◽

Cellular Processes ◽

Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

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