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Methods Protoc., Volume 1, Issue 1 (December 2017)

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Editorial

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Open AccessEditorial Welcome to the New Journal Methods and Protocols
Methods Protoc. 2017, 1(1), 1; doi:10.3390/mps1010001
Received: 21 June 2017 / Revised: 21 June 2017 / Accepted: 21 June 2017 / Published: 27 June 2017
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Abstract
I am pleased to introduce Methods and Protocols, a new multidisciplinary, peer-reviewed, open access journal devoted to providing an open forum to report on new procedural approaches and cutting-edge methodological developments.[...] Full article

Research

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Open AccessArticle Predictable Peptide Conjugation Ratios by Activation of Proteins with Succinimidyl Iodoacetate (SIA)
Methods Protoc. 2017, 1(1), 2; doi:10.3390/mps1010002
Received: 2 August 2017 / Revised: 12 September 2017 / Accepted: 14 September 2017 / Published: 25 September 2017
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Abstract
The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide–protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as
[...] Read more.
The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide–protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as Traut’s reagent. Both approaches are difficult to control, need individual optimization and often lead to unsatisfactory results. In another popular approach, a heterobifunctional linker with a N-hydroxysuccinimide (NHS) and a maleimide functionality is applied to the protein. After the activation of some lysine ε-amino groups with the NHS ester functionality, a cysteine-containing peptide is attached to the activated carrier protein via maleimide. Particularly, the maleimide reaction leads to some unwanted byproducts or even cleavage of the linker. Many protocols end up with conjugates with unpredictable and irreproducible conjugation ratios. In addition, the maleimide-thiol addition product should be assumed immunogenic in vivo. To avoid these and other disadvantages of the maleimide approach, we examined the known linker succinimidyl iodoacetate (SIA) in more detail and developed two protocols, which lead to peptide–protein conjugates with predefined average conjugation ratios. This holds potential to eliminate tedious and expensive optimization steps for the synthesis of a bioconjugate of optimal composition. Full article
(This article belongs to the Special Issue Feature Papers)
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Open AccessArticle The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol
Methods Protoc. 2017, 1(1), 4; doi:10.3390/mps1010004
Received: 31 October 2017 / Revised: 26 November 2017 / Accepted: 27 November 2017 / Published: 1 December 2017
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Abstract
A single solution protocol has been widely used for the fluorimetric determination of H2O2 in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin
[...] Read more.
A single solution protocol has been widely used for the fluorimetric determination of H2O2 in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin and H2O2 in the sample and the subsequent internal additions, and the measurements of the fluorescence are all carried out at a single pH in a fluorometer cell. It is found that this protocol is prone to four sources of possible error. The variability in the reaction stoichiometry between scopoletin and H2O2 in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H2O2, the photobleaching of scopoletin, and the de-activation of HRP. These possible sources of error can be circumvented in a two-stage protocol in which the reaction between H2O2 and scopoletin is carried out immediately upon sampling at a pH of 7, and the measurement of the fluorescence is carried out later on at a pH of 9. It should be the protocol of choice. Furthermore, in the two-stage protocol, after the initial reaction between H2O2 and scopoletin, the sample may be stored at room temperature for six days and at 4 °C for at least a month before its fluorescence is measured. This option can significantly reduce the logistics in the field. Full article
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Other

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Open AccessBenchmark A Highly Sensitive Non-Radioactive Activity Assay for AMP-Activated Protein Kinase (AMPK)
Methods Protoc. 2017, 1(1), 3; doi:10.3390/mps1010003
Received: 15 August 2017 / Revised: 26 September 2017 / Accepted: 6 October 2017 / Published: 13 October 2017
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Abstract
While many methods exist to quantitatively determine protein kinase activities, 32P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we
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While many methods exist to quantitatively determine protein kinase activities, 32P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for phosphorylation by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation sensors, can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio. Full article
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