Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time‐saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC.
Studying and quantifying the mechanics of blood clots is essential to better diagnosis and prognosis of, as well as therapy for, thromboembolic pathologies such as strokes, heart attacks, and pulmonary embolisms. Unfortunately, mechanically testing blood clots is complicated by their softness and fragility, thus making the use of classic mounting techniques, such as clamping, challenging. This is particularly true for mechanical testing under large deformation. Here, we describe protocols for creating in vitro blood clots and securely mounting these samples on mechanical test equipment. To this end, we line 3D‐printed molds with a hook‐and‐loop fabric that, after coagulation, provides a secure interface between the sample and device mount. In summary, our molding and mounting protocols are ideal for performing large‐deformation mechanical testing, with samples that can withstand substantial deformation without delaminating from the apparatus. © 2021 Wiley Periodicals LLC.
- NSF-PAR ID:
- 10276768
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 7
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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This article was corrected on 26 June 2021. See the end of the full text for details.
Basic Protocol 1 : Total RNA isolation from camelid leukocytesBasic Protocol 2 : First‐strand cDNA synthesis; VHH and VHrepertoire PCRBasic Protocol 3 : Preparation of the phage display libraryBasic Protocol 4 : Panning of the phage display libraryBasic Protocol 5 : Small‐scale nAb expressionBasic Protocol 6 : Sequence analysis of selected nAb clonesBasic Protocol 7 : Nanobody validation as immunolabelsBasic Protocol 8 : Generation of nAb‐pEGFP mammalian expression constructsBasic Protocol 9 : Nanobody validation as intrabodiesSupport Protocol 1 : ELISA for llama serum testing, phage titer, and screening of selected clonesSupport Protocol 2 : Amplification of helper phage stockSupport Protocol 3 : nAb expression in amber suppressorE. coli bacterial strains -
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Basic Protocol 1 : Harvesting and dissection of the joint surfacesBasic Protocol 2 : Preparation of samples for microindentation and fatigue testingBasic Protocol 3 : Microfracture using microindentationBasic Protocol 4 : Crack propagation under cyclic loading -
Abstract Glycosaminoglycans (GAGs) are linear polysaccharides found in a variety of organisms. GAGs contribute to biochemical pathway regulation, cell signaling, and disease progression. GAG sequence information is imperative for determining structure‐function relationships. Recent advances in electron‐activation techniques paired with high‐resolution mass spectrometry allow for full sequencing of GAG structures. Electron detachment dissociation (EDD) and negative electron transfer dissociation (NETD) are two electron‐activation methods that have been utilized for GAG characterization. Both methods produce an abundance of informative glycosidic and cross‐ring fragment ions without producing a high degree of sulfate decomposition. Here, we provide detailed protocols for using EDD and NETD to sequence GAG chains. In addition to protocols directly involving performing these MS/MS methods, protocols include sample preparation, method development, internal calibration, and data analysis. © 2021 Wiley Periodicals LLC.
This article was corrected on 27 July 2022. See the end of the full text for details.
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