FPCount protocol - in-lysate (purification free) protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Quantification ◽  
Protein Storage ◽  
Cell Lysates ◽  
Concentration Determination ◽  
His Tag ◽  
Plate Reader ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression and production of cell lysates. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This in-lysate version of the protocol uses the ECmax protein quantification protocol of FPs in lysates and does not require His-tag purification of the FPs. Note that it is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the 'short' protocol, which requires FP purification, or the 'complete' protocol, which requires FP purification and compares three protein quantification methods. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - Short protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Quantification ◽  
Short Version ◽  
Protein Storage ◽  
Concentration Determination ◽  
Sds Page ◽  
Plate Reader ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This short version uses the ECmax protein quantification protocol, and is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the complete protocol, which describes three protein quantification methods. The short protocol also skips the SDS-PAGE steps. If you require these, please see the complete protocol. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 6. Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - Full protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Storage ◽  
Concentration Determination ◽  
Plate Reader ◽  
Full Protocol ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 5. Gel1: Verification of Expression/Fractions 6. Purification 7. Gel2: Verification of Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 11. Quantification of FP concentration (part2) 12. Protein Storage 13. Calibration of Plate Reader

Targeting and processing of membrane-anchored YFP fusion proteins to protein storage vacuoles in transgenic tobacco seeds

2005 ◽  
Vol 15 (4) ◽  
pp. 361-364 ◽  
Author(s):  
Ka Leung Fung ◽  
Yin Fun Yim ◽  
Yu Chung Tse ◽  
Yansong Miao ◽  
Samuel S.M. Sun ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Fusion Proteins ◽  
Seed Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Pharmaceutical Proteins ◽  
Tobacco Seeds ◽  
Foreign Proteins

Seeds that store proteins in protein storage vacuoles are attractive bioreactors for producing and storing large amounts of pharmaceutical proteins. However, foreign proteins expressed in transgenic plants are subjected to the delivery and modification processes present within plant cells. Here, it is demonstrated that unique membrane sequences deliver a yellow fluorescent protein (YFP) to the seed protein storage vacuoles in transgenic tobacco (Nicotiana tabacum L.) plants, where the YFP is then separated from its membrane anchors. This precise targeting and separation is required for the successful delivery of useful proteins to seed protein storage vacuoles for their stable accumulation in transgenic crops.

scholarly journals Comparison of two methods of tear sampling for protein quantification by Bradford method

2013 ◽  
Vol 33 (2) ◽  
pp. 261-264 ◽  
Author(s):  
Eliana Farias ◽  
Katia L. Yasunaga ◽  
Romulo V.R. Peixoto ◽  
Micaella P. Fonseca ◽  
Wagner Fontes ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Protein Concentration ◽  
Protein Pattern ◽  
Protein Quantification ◽  
Tear Protein ◽  
Schirmer Tear Test ◽  
Healthy Dogs ◽  
Bradford Method ◽  
Student’S T ◽  
Student’S T Test

The aim of this study was to compare two methods of tear sampling for protein quantification. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80ºC and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

scholarly journals A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid

2021 ◽  
Author(s):  
Danielle M. Stevens ◽  
Andrea Tang ◽  
Gitta Coaker
Keyword(s):  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Region Of Interest ◽  
R Package ◽  
Clavibacter Michiganensis ◽  
Gram Positive ◽  
Integrative Plasmid ◽  
Tomato Pathogen ◽  
Green Fluorescent ◽  
Markerless Deletion

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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