Table of Contents

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
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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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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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