Selection of appropriate protein assay method for a paper microfluidics platform

CRISPR-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach and it can generate unintended transgenic integrations, while Cas9 prolonged expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated cells, especially plant cells, is still challenging. These downfalls can be avoided with the delivery of preassembled ribonucleoprotein complexes (RNPs) composed of purified recombinant enzyme Cas9 and in vitro- transcribed guide RNA (gRNA) molecules in a protoplast system. We therefore aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with PEG 4000. We performed effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene and applying two different exposure times to RNPs. Using low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between exposure time and mutation frequency. Mutation frequency was gRNA sequence- and exposure time-dependent. In summary, we demonstrated the suitability of RNP transfection as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for selection of gRNA suitable for editing of gene of interest will be highly useful for genome editing in maize, since genome size and GC-content are large and high in maize genome, respectively. Nevertheless, the large amplitude of mutations at target site requires scrutiny when checking mutations at off-target sites and potential safety concerns.

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A Novel Protein Assay Method Using Tetraphenylporphin tetrasulfonate (TPPS4)

Analytical Letters ◽

10.1080/00032719508000354 ◽

1995 ◽

Vol 28 (10) ◽

pp. 1763-1774 ◽

Cited By ~ 21

Author(s):

N. Li ◽

K. A. Li ◽

S. Y. Tong

Keyword(s):

Assay Method ◽

Protein Assay ◽

Novel Protein

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The Optimization of Synthesis Condition of ZnSe Nanocrystals

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.534.61 ◽

2012 ◽

Vol 534 ◽

pp. 61-64

Author(s):

Ting Wang ◽

Li Guo

Keyword(s):

Protein Concentration ◽

Self Organization ◽

Assay Method ◽

Tween 20 ◽

Functional Material ◽

Protein Assay ◽

Protein Cages ◽

The Core ◽

Znse Nanocrystals ◽

Absorption Measurements

In biomineralization, self-organization of organic based templates provides scaffolding for the assembly of QDs materials. The host-guest relationship between these protein cages and the encapsulated material is based primarily on a complementary electrostatic interaction. Zinc selenide (ZnSe) were synthesized in the cavity of the apoferritin from horse spleen (HsAFr) and the reaction condition was optimized by adding tween 20 to avoid ferritin agglomeration. The obtained nanodots were characterized by TEM, and absorption measurements. In addition, the protein concentration of ZnSe-ferritin was precisely measured by the Bradford protein assay method. From the results, it was concluded that the ZnSe nanocrystals were successfully synthesized in the core of ferritin and it can be applied as a potential functional material such as transistors, biosensor materials or medical imaging.

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Rapid Fluorescein and Protein Assay Method for Fluorescent-Antibody Conjugates

Applied Microbiology ◽

10.1128/aem.14.2.271-275.1966 ◽

1966 ◽

Vol 14 (2) ◽

pp. 271-275 ◽

Cited By ~ 89

Author(s):

Arthur F. Wells ◽

Curtis E. Miller ◽

Marvin K. Nadel

Keyword(s):

Fluorescent Antibody ◽

Assay Method ◽

Protein Assay ◽

Antibody Conjugates

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Anti Xa Monitoring during Treatment with Low Molecular Weight Heparin or Danaparoid: Inter-assay Variability

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614377 ◽

1999 ◽

Vol 82 (10) ◽

pp. 1289-1293 ◽

Cited By ~ 38

Author(s):

R. Iampietro ◽

A. M. Woolley ◽

F. E. Preston ◽

S. Kitchen

Keyword(s):

Molecular Weight ◽

Therapeutic Range ◽

Low Molecular Weight Heparin ◽

Assay Method ◽

Target Range ◽

Low Molecular Weight ◽

The Mean ◽

The Relationship ◽

Higher Doses ◽

Selection Of

SummaryIf laboratory monitoring of low molecular weight heparin (LMWH) therapy is required the test of choice is the anti Xa activity assay. The relationship between anti Xa results obtained using different techniques is unknown. The aim of the present study was to compare anti Xa results obtained with eight different commercially available anti Xa activity assays (five chromogenic and three clotting based assays) in samples from patients receiving either therapeutic or prophylactic LMWH (enoxaparin or dalteparin) or danaparoid.We have demonstrated that highly significant differences exist between results obtained using different techniques. The mean anti Xa activity in patients receiving treatment or prophylaxis with enoxaparin ranged from 0.28 to 0.64 iu/ml. A similar relationship was present in samples from patients treated with dalteparin, mean anti Xa results ranging from 0.43 to 0.69 iu/ml. The Heptest clotting assay as used here in combination with the Automated Coagulation Laboratory instrument, was associated with lower results than other clotting or chromogenic techniques. In patients receiving danaparoid for heparin induced thrombocytopaenia (HIT) mean results with three clotting based assays were 0.30 to 0.36 u/ml, compared to mean results of 0.47 to 0.65 u/ml for chromogenic assays.Our data clearly indicate that the selection of anti Xa assay method could influence patient management since the dose required to achieve the therapeutic range would differ according to the assay employed. This is particularly important since the frequency of hamorrhagic side effects has been shown by others to be dose dependant, irrespective of the concomitant anti Xa activity results. In danaparoid therapy the clotting assays studied here should not be employed for monitoring without a modified target range, unless it can be demonstrated that the higher doses required to achieve the therapeutic range are safe.

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PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Genes ◽

10.3390/genes11091029 ◽

2020 ◽

Vol 11 (9) ◽

pp. 1029 ◽

Cited By ~ 2

Author(s):

Rodrigo Ribeiro Arnt Sant’Ana ◽

Clarissa Alves Caprestano ◽

Rubens Onofre Nodari ◽

Sarah Zanon Agapito-Tenfen

Keyword(s):

Exposure Time ◽

Dna Sequences ◽

Mutation Frequency ◽

Gene Editing ◽

Inositol Phosphate ◽

Gc Content ◽

Assay Method ◽

Ribonucleoprotein Complexes ◽

Target Sites ◽

Selection Of

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

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Calibrator material selection: a key criteria during biomarker assay method development

Bioanalysis ◽

10.4155/bio-2020-0208 ◽

2021 ◽

Author(s):

Sai P Thankamony ◽

Rong Liu ◽

Jon E Peterson ◽

Rasa Santockyte ◽

Timothy Olah ◽

...

Keyword(s):

Decision Making ◽

Case Studies ◽

Method Development ◽

Material Selection ◽

Assay Method ◽

Context Of Use ◽

The Impact ◽

Selection Of

Biomarker assay method development is a multistep rigorous process and calibrant material selection is integral to ensuring the quality of such assays. However, the impact of selection of calibrator material may often get overlooked. In this article, we highlight three case studies where biomarker calibrant material selection was deemed an essential criterion for consideration. Through these case studies we highlight challenges faced, steps taken and discuss the impact on assay-related decision-making. We also provide additional perspectives for selection and characterization of calibrant proteins in the setting of an evolving biomarker context of use.

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