scholarly journals Chloroform-Methanol Protein Extraction with Zymolyase Treatment for Yeast (High Throughput) v1 (protocols.io.bhptj5nn)

protocols.io ◽  
2020 ◽  
Author(s):  
Jennifer Gin ◽  
Yan Chen ◽  
Christopher Petzold
2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System


Author(s):  
Ajay Iyer ◽  
Lisa Guerrier ◽  
Salomé Leveque ◽  
Charles S. Bestwick ◽  
Sylvia H. Duncan ◽  
...  

AbstractInvasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.


2020 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System


2022 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system


2019 ◽  
Vol 2 (1) ◽  
pp. 15 ◽  
Author(s):  
Martin Broberg ◽  
James E. McDonald

The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota.


2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Tad Ogorzalek ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System


2021 ◽  
Author(s):  
Yan Chen ◽  
Tad Ogorzalek ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Christopher J Petzold

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system


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