Recent advances in microscale western blotting

Sanders, Brittany J.; Kim, Daniel C.; Dunn, Robert C.

doi:10.1039/c6ay01947a

Cited by 20 publications

(11 citation statements)

References 99 publications

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“…WB could also be used for quantitative determinations 44 . Furthermore, in light of more recent miniaturization of the technique such as WB in chip format, the technique could sustain importance for future application 45 even though most blotting investigations are anticipated to be replaced by MS.…”

Section: Western Blotmentioning

confidence: 99%

Instant on-paper protein digestion during blood spot sampling

Skjærvø

Rosting

Halvorsen

et al. 2017

Analyst

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A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

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Section: Western Blotmentioning

confidence: 99%

Instant on-paper protein digestion during blood spot sampling

Skjærvø

Rosting

Halvorsen

et al. 2017

Analyst

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“…Although various new and improved proteomic techniques are available today, traditional gel‐based proteomics, such as western blotting and two‐dimensional electrophoresis, are still widely used (Chandrasekhar, Dileep, Lebonah, & Kumari, ; Magdeldin et al, ). Western blotting is a semi‐quantitative technique used extensively in research to detect specific proteins and determine their levels (Sanders, Kim, & Dunn, ). Proteins in crude samples are separated based on size using gel electrophoresis, transferred to membranes, and detected by antibodies specific for the protein of interest (Sanders et al, ).…”

Section: Aptamer Selection Using the Protein‐transferred Membrane Aftmentioning

confidence: 99%

“…Western blotting is a semi‐quantitative technique used extensively in research to detect specific proteins and determine their levels (Sanders, Kim, & Dunn, ). Proteins in crude samples are separated based on size using gel electrophoresis, transferred to membranes, and detected by antibodies specific for the protein of interest (Sanders et al, ).…”

Section: Aptamer Selection Using the Protein‐transferred Membrane Aftmentioning

confidence: 99%

“…Although various new and improved proteomic techniques are available today, traditional gelbased proteomics, such as western blotting and two-dimensional electrophoresis, are still widely used (Chandrasekhar, Dileep, Lebonah, & Kumari, 2014;Magdeldin et al, 2014). Western blotting is a semiquantitative technique used extensively in research to detect specific proteins and determine their levels (Sanders, Kim, & Dunn, 2016).…”

Section: Aptamer Selection Using the Protein-transferred Membrane Amentioning

confidence: 99%

“…Proteins in crude samples are separated based on size using gel electrophoresis, transferred to membranes, and detected by antibodies specific for the protein of interest (Sanders et al, 2016). 4.1 | Aptamer selection against proteins immobilized on membrane after SDS-PAGE Bianchini et al (2001) originally developed aptamer selection on a protein-transfer membrane.…”

Section: Aptamer Selection Using the Protein-transferred Membrane Amentioning

confidence: 99%

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Development of aptamers against unpurified proteins

Goto

Tsukakoshi

2017

Biotech & Bioengineering

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SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples.

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Western Blotting with Solutions containing Nanoliter Volumes of Antibody

Walma

Collins

2019

CP Cell Biology

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Whether screening small mammal serum during antibody production or attempting to preserve a stock of precious antibody, this protocol's western blotting method using aliquots containing nanoliter volumes of antibody will benefit researchers. Time‐tested western blotting workflows allowing separation and analysis of proteins are routinely utilized in clinical and laboratory settings. The necessity for relatively large quantities of antibody is a major limitation to this universal tool. This article provides a step‐by‐step protocol for detecting proteins of interest with solutions containing nanoliter volumes of antibody without altering the preceding gel electrophoresis and transfer methods. Important considerations, frequently encountered problems, and means of optimizing reproducibility are discussed. Complementary diagrams, images, and videos are provided. The protocol is demonstrated using 0.3 nanoliters of anti‐serum to detect fibronectin in a human foreskin fibroblast cell line. Finally, two support protocols detailing methods of extracting proteins from cultured cells are reported. Published 2019. This article is a US Government work and is in the public domain in the USA.

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Recent advances in microscale western blotting

Cited by 20 publications

References 99 publications

Instant on-paper protein digestion during blood spot sampling

Instant on-paper protein digestion during blood spot sampling

Development of aptamers against unpurified proteins

Western Blotting with Solutions containing Nanoliter Volumes of Antibody

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