One-step analysis of protein complexes in microliters of cell lysate

2005 ◽  
Vol 2 (11) ◽  
pp. 833-835 ◽  
Author(s):  
Oda Stoevesandt ◽  
Karsten Köhler ◽  
Rainer Fischer ◽  
Ian C D Johnston ◽  
Roland Brock
2016 ◽  
Vol 200 ◽  
pp. 90-101 ◽  
Author(s):  
Amenehsadat Hashemi ◽  
Javad Gharechahi ◽  
Ghorbanali Nematzadeh ◽  
Faezeh Shekari ◽  
Seyed Abdollah Hosseini ◽  
...  

2019 ◽  
Author(s):  
Deepanksha Arora ◽  
Nikolaj B. Abel ◽  
Chen Liu ◽  
Petra Van Damme ◽  
Klaas Yperman ◽  
...  

AbstractProximity-dependent biotin labelling (PDL) uses a promiscuous biotin ligase (PBL) or a peroxidase fused to a protein of interest. This enables covalent biotin labelling of proteins and allows subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact. To date, only few papers report on the use of PDL in plants. Here we present the results of a systematic study applying a variety of PDL approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols which combine Mass Spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as test-case. We further benchmark the efficiency of various PBLs in comparison with one-step affinity purification approaches. We identified both known as well as novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both non-biotinylated as well as biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to infer structural information of protein complexes.


2007 ◽  
Vol 1151 (1-2) ◽  
pp. 158-163 ◽  
Author(s):  
Yoichi Shibusawa ◽  
Naoko Takeuchi ◽  
Kanako Tsutsumi ◽  
Shigeru Nakano ◽  
Akio Yanagida ◽  
...  

1995 ◽  
Vol 13 (2) ◽  
pp. 155-160 ◽  
Author(s):  
Gerald Kleymann ◽  
Christian Ostermeier ◽  
Bernd Ludwig ◽  
Arne Skerra ◽  
Hartmut Michel

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Célia Arib ◽  
Jolanda Spadavecchia ◽  
Marc Lamy de la Chapelle

AbstractLarge protein complexes carry out some of the most complex activities in biology1,2. Such structures are often assembled spontaneously through the process of self-assembly and have characteristic chemical or biological assets in the cellular mechanisms3. Gold-based nanomaterials have attracted much attention in many areas of chemistry, physics and biosciences because of their size- and shape-dependent optic, electric, and catalytic properties. Here we report for the first time a one step synthesis in which Manganese Superoxide Dismutase protein plays a key role in the reduction of gold salts via the use of a Good's buffer (HEPES) to produce gold nanoparticles, compared to other proteins as catalase (CAT) and bovine serum albumin (BSA).We prove that this effect is directly related with the biological activities of the proteins that have an effect on the gold reduction mechanisms. Such synthesis route also induces the integration of proteins directly in the AuNPs that are intrinsically safe by design using a one-step production method. This is an important finding that will have uses in various applications, particularly in the green synthesis of novel nanomaterials.


2019 ◽  
Author(s):  
Claudio Schmidli ◽  
Stefan Albiez ◽  
Luca Rima ◽  
Ricardo Righetto ◽  
Inayatulla Mohammed ◽  
...  

AbstractHigh-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.


2019 ◽  
Vol 116 (30) ◽  
pp. 15007-15012 ◽  
Author(s):  
Claudio Schmidli ◽  
Stefan Albiez ◽  
Luca Rima ◽  
Ricardo Righetto ◽  
Inayatulla Mohammed ◽  
...  

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.


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