scholarly journals One‐pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds

2019 ◽  
Author(s):  
Cuimei Liu ◽  
Dongmei Li ◽  
Jincheng Li ◽  
Zhenpeng Guo ◽  
Yi Chen
Keyword(s):  
Spatial Distribution ◽  
Sample Preparation ◽  
One Pot ◽  
Single Shoot

SEM evaluation of the TEM cold-stage double-film specimen

1984 ◽  
Vol 42 ◽  
pp. 272-273
Author(s):  
Jayesh Bellare
Keyword(s):  
Spatial Distribution ◽  
Sample Preparation ◽  
Sample Thickness ◽  
Specimen Preparation ◽  
Preparation Technique ◽  
Liquid Sample ◽  
Thin Specimen ◽  
Sample Preparation Technique ◽  
Cold Stage ◽  
Film Specimen

Seeing is believing, but only after the sample preparation technique has received a systematic study and a full record is made of the treatment the sample gets.For microstructured liquids and suspensions, fast-freeze thermal fixation and cold-stage microscopy is perhaps the least artifact-laden technique. In the double-film specimen preparation technique, a layer of liquid sample is trapped between 100- and 400-mesh polymer (polyimide, PI) coated grids. Blotting against filter paper drains excess liquid and provides a thin specimen, which is fast-frozen by plunging into liquid nitrogen. This frozen sandwich (Fig. 1) is mounted in a cooling holder and viewed in TEM.Though extremely promising for visualization of liquid microstructures, this double-film technique suffers from a) ireproducibility and nonuniformity of sample thickness, b) low yield of imageable grid squares and c) nonuniform spatial distribution of particulates, which results in fewer being imaged.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Katharina Habler ◽  
Bernhard Koeppl ◽  
Franz Bracher ◽  
Michael Vogeser
Keyword(s):  
Sample Preparation ◽  
Bile Acids ◽  
High Performance ◽  
Solid Phase ◽  
Sulfate Solution ◽  
Two Dimensional ◽  
Bioanalytical Method Validation ◽  
One Pot ◽  
Internal Standards ◽  
Secondary Bile Acids

Abstract Objectives Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Methods Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. Results The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) “Guideline on bioanalytical method validation”. To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Conclusions Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

scholarly journals Sample Preparation by Easy Extraction and Digestion (SPEED) - A Universal, Rapid, and Detergent-free Protocol for Proteomics based on Acid Extraction

2018 ◽  
Author(s):  
Joerg Doellinger ◽  
Andy Schneider ◽  
Marcell Hoeller ◽  
Peter Lasch
Keyword(s):  
Sample Preparation ◽  
Protein Extraction ◽  
Spectrometric Analysis ◽  
Acid Extraction ◽  
Sample Processing ◽  
Special Equipment ◽  
One Pot ◽  
Tissue Samples ◽  
Main Challenge ◽  
Speed Performance

SUMMARYMass spectrometry is the method of choice for deep and comprehensive analysis of proteomes and has become a key technology to support the progress in life science and biomedicine. However, sample preparation in proteomics is not standardized and contributes to a lack of reproducibility. The main challenge is to extract all proteins in a manner that enables efficient digestion into peptides and is compatible with subsequent mass spectrometric analysis. Current methods are based on the idea of removing detergents or chaotropic agents during sample processing, which are essential for protein extraction but interfere with digestion and LC-MS. These multi-step preparations are prone to losses, biases and contaminations, while being time-consuming and labor-intensive.We report a universal detergent-free method, named Sample Preparation by Easy Extraction and Digestion (SPEED), which is based on a simple three-step procedure, acidification, neutralization and digestion. SPEED is a one-pot method for peptide generation from various sources and is easily applicable even for lysis-resistant sample types as pure trifluoroacetic acid (TFA) is used for highly efficient protein extraction. SPEED-based sample processing is highly reproducible, provides exceptional peptide yields and enables preparation even of tissue samples with less than 15 min hands-on time and without any special equipment. Evaluation of SPEED performance revealed, that the number of quantified proteins and the quantitative reproducibility are superior compared to the well-established sample processing protocols FASP, ISD-Urea and SP3 for various sample types, including human cells, bacteria and tissue, even at low protein starting amounts.

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells v1

protocols.io ◽  
2021 ◽  
Author(s):  
Kendall not provided Martin ◽  
Tong not provided Zhang ◽  
William not provided B. Chrisler ◽  
Fillmore not provided L. Thomas ◽  
Wei-Jun not provided Qian ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Human Cells ◽  
Targeted Proteomics ◽  
Proteomics Analysis ◽  
One Pot

A one-pot, isothermal DNA sample preparation and amplification platform utilizing aqueous two-phase systems

2018 ◽  
Vol 410 (21) ◽  
pp. 5255-5263 ◽  
Author(s):  
Sherine F. Cheung ◽  
Matthew F. Yee ◽  
Nguyen K. Le ◽  
Benjamin M. Wu ◽  
Daniel T. Kamei
Keyword(s):  
Sample Preparation ◽  
Two Phase ◽  
One Pot ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

scholarly journals Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Chia-Feng Tsai ◽  
Pengfei Zhang ◽  
David Scholten ◽  
Kendall Martin ◽  
Yi-Ting Wang ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Single Cell ◽  
Tumor Cells ◽  
Cancer Metastasis ◽  
Single Cells ◽  
Surface Adsorption ◽  
Detection Sensitivity ◽  
Label Free ◽  
One Pot ◽  
Surfactant Assisted

AbstractLarge numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

High-Resolution Chemical Mapping and Identifying Spatial Distribution of Microbes in the Zea mays Rhizosphere using Correlative Microscopy

2021 ◽  
Author(s):  
Chiththaka Chaturanga D B Imihami Mudiyanselage ◽  
Matthias Schmidt ◽  
Yalda Davoudpour ◽  
Hryhoriy Stryhanyuk ◽  
Hans Richnow ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Sample Preparation ◽  
Soil Chemistry ◽  
Preparation Method ◽  
Chemical Mapping ◽  
Sample Preparation Method ◽  
Water Jet Cutting ◽  
Card Fish ◽  
Rhizosphere Processes

<p>Studying the spatial distribution of bacteria and characterizing the soil chemistry (i.e., elemental, isotopic and molecular composition) underpins the comprehensive understanding of rhizosphere associated processes. During the past decades, several stand-alone methods have been developed to investigate soil chemistry, nutrient cycling and plant nutrition. However, individual approaches as stand-alone are not capable of providing an overall rhizosphere processes involving soil, root and microbes in a spatial context, as there is no common sample preparation method available to satisfy individual needs of each technique. Here, we present i) a sample preparation method, which includes soil embedding, sectioning and ii) a correlative imaging and image registration workflow, which allows for characterization of minerals, roots and microbes by different high-resolution imaging and microanalytical techniques. This allows for conducting rhizosphere studies on different scales, focusing on root-soil-microbe interfaces with spatial resolution of nano-meter scale. Hydrophilic, immunohistochemistry compatible, low viscosity LR White resin was used to embed and stabilize the soil and make it ultra-high vacuum compatible. We employed water-jet cutting as a novel approach to slice the embedded samples, and, by doing so, avoided polishing of the surface which often leads to translocation of sample material (smearing). The quality of this embedding was analyzed by and Helium Ion Microscopy (HIM). Epifluorescence microscopy in combination with Catalyzed Reporter Deposition-Fluorescence in-situ Hybridization (CARD-FISH) was employed to accurately identify and determine the spatial distribution of bacteria in the embedded sample, thus avoiding ambiguities from high levels of auto-fluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was acquired by SEM/EDX, ToF-SIMS, nanoSIMS for elemental, molecular and isotopic characterization, respectively, and µ-Raman microscopy for specific identification of minerals.</p><p>In summary, we demonstrate that LR White embedding and water-jet cutting of soil in combination with CARD-FISH and a correlative microscopic workflow, allows for a comprehensive characterization of biotic and abiotic components in the rhizosphere. The developed sample preparation method can facilitate the various requirements of involved microscopy techniques and individual workflows for imaging and image registration to analyze data. We foresee that this approach will establish an excellent platform to study various soil processes and synergistic understanding of complex rhizosphere processes.</p>

scholarly journals Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

10.3791/61797 ◽  
2020 ◽  
Author(s):  
Kendall Martin ◽  
Tong Zhang ◽  
Pengfei Zhang ◽  
William B. Chrisler ◽  
Fillmore L. Thomas ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Human Cells ◽  
Targeted Proteomics ◽  
Proteomics Analysis ◽  
One Pot

scholarly journals All-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay protocol for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Xiong Ding ◽  
Kun Yin ◽  
Ziyue Li ◽  
Rajesh V. Lalla ◽  
Maroun M. Sfeir ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Fluorescence Detection ◽  
Visual Detection ◽  
Imaging System ◽  
Point Of Care ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
One Pot ◽  
Reaction Systems ◽  
Assay Protocol

Abstract This protocol presents the all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay to ultra-sensitively and visually detect SARS-CoV-2. The procedure of AIOD-CRISPR assay typically consists of three parts including sample preparation, AIOD-CRISPR reaction, and fluorescence detection. Sample preparation involves the synthetic RNA preparation and the nucleic acid extraction from SARS-CoV-2 samples. The prepared nucleic acids were then added into the AIOD-CRISPR reaction systems as templates, followed by incubation at 37°C for 20-40 min. After incubation, visual detection was immediately conducted by placing the tubes in a portable LED blue transilluminator (Maestrogen UltraSlim) or the ChemiDoc™ MP Imaging System (Bio-Rad) with its built-in UV channel. In addition to endpoint visual detection, real-time fluorescence detection was also available for AIOD-CRISPR assay. This protocol is helpful for applying AIOD-CRISPR assay to rapid, sensitive, one-pot point-of-care SARS-CoV-2 detection.

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