| 41. |
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| 42. |
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| 43. |
- Birgersson, Madeleine, et al.
(författare)
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Antibody Validation for Estrogen Receptor Beta
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 1-23, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.
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| 44. |
- Björkbacka, Harry
(författare)
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Microarray Experiments to Uncover Toll-Like Receptor Function.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 517, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- This chapter is intended as a handbook for anyone interested in using microarrays to study Toll-like receptor (TLR) function or any other biological question. Although microarray technology has developed into a standard tool at many laboratories disposal, most of the actual microarray processing is done by core facilities using highly specialized equipment. This chapter only briefly describes these methods in principle and instead focus on the parts that investigators themselves can influence, such as the experimental design, RNA isolation, statistical analysis, cluster analysis, data visualization, and biological interpretation.
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| 45. |
- Björklund, Tomas
(författare)
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Expression of Multiple Functional RNAs or Proteins from One Viral Vector.
- 2016
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029. ; 1382, s. 41-56
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Tidskriftsartikel (refereegranskat)abstract
- In this chapter, we will cover the available design choices for enabling expression of two functional protein or RNA sequences from a single viral vector. Such vectors are very useful in the neuroscience-related field of neuronal control and modulation, e.g., using optogenetics or DREADDs, but are also desirable in applications of CRISPR/Cas9 in situ genome editing and more refined therapeutic approaches. Each approach to achieving this combined expression has its own strengths and limitations, which makes them more or less suitable for different applications. In this chapter, we describe the available alternatives and provide tips on how they can be implemented.
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| 46. |
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| 47. |
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| 48. |
- Bonander, Nicklas, 1968, et al.
(författare)
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Optimising yeast as a host for recombinant protein production (review)
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. ; 866, s. 1-9
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Forskningsöversikt (refereegranskat)abstract
- Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.
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| 49. |
- Borrebaeck, Carl A K, et al.
(författare)
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Recombinant Antibody Microarray for Profiling the Serum Proteome of SLE.
- 2014
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029. ; 1134, s. 67-78
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Tidskriftsartikel (refereegranskat)abstract
- Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are deposited one by one in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified. The binding pattern is then converted into a relative protein expression map, or protein map, deciphering the composition of the sample at the molecular level. The methodology provides unique opportunities for delineating serum biomarkers reflecting SLE, thus paving the way for improved diagnosis, classification, and prognosis.
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| 50. |
- Borrebaeck, Carl, et al.
(författare)
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Antibody array generation and use.
- 2014
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 1131, s. 563-571
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteomics, represented by antibody arrays, is a multiplex technology for high-throughput protein expression profiling of crude proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are individually deposited in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified using mainly fluorescence as the mode of detection. The binding pattern is then converted into a relative protein expression map, or protein atlas, delineating the composition of the sample at the molecular level. The technology provides unique opportunities for various applications, such as protein expression profiling, biomarker discovery, disease diagnostics, prognostics, evidence-based therapy selection, and disease monitoring. Here, we describe the generation and use of planar antibody arrays for serum protein profiling.
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