Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging

Zhang, Ning; Chen, Jinlong

doi:10.1002/0471142727.mb1015s41

Cited by 4 publications

(5 citation statements)

References 15 publications

(17 reference statements)

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“…A frequently used option is to add a short peptide or epitope that is recognized by a commercially available high‐affinity monoclonal antibody (MAb; Zhang and Chen, ). The epitope is typically added at the amino or carboxyl terminus, although internal positions that do not disrupt function can also be used.…”

Section: Strategic Planningmentioning

confidence: 99%

“…For additional background reading, the user should consult UNIT here for a theoretical discussion and methods for immunoprecipitation; see Chapter 2 for principles of antibody production and immunoassays. For an in‐depth review of protein tagging, see Zhang and Chen (); see Treco and Winston () for transformation and propagation of S. cerevisiae . For an in‐depth review of coprecipitation and other approaches to detect protein‐protein interactions, see Phizicky and Fields (), Toby and Golemis (), Auerbach et al (), Gavin and Superti‐Ferga (), and Kerppola ().…”

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confidence: 99%

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Detection of Protein‐Protein Interactions by Coprecipitation

Elion

2007

CP in Immunology

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Coprecipitation of proteins from whole-cell extracts is a valuable approach to test for physical interactions between proteins of interest. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The approaches described can be adapted for other systems. In a typical experiment, as described here, cells are lysed and a whole-cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid-phase affinity matrix, and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high affinity to a molecule that can be coupled to a solid-phase matrix.

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Section: Strategic Planningmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of Protein‐Protein Interactions by Coprecipitation

Elion

2007

CP in Immunology

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“…For instance, epitope tags can be used as an affinity tag to perform protein purification when using stringent purification conditions or to identify interacting proteins when combining non-stringent affinity purifications and mass spectrometry analysis of co-purified proteins [16-27]. Moreover, modifications of the small peptide sequence may further enlarge the range of applications [28].…”

Section: Discussionmentioning

confidence: 99%

Immunohistochemical detection of transgene expression in the brain using small epitope tags

et al. 2010

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BackgroundIn vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags.ResultsIn the present study, we evaluated several small epitope tags together with corresponding anti-tag antibodies for the detection of overexpressed proteins in rat brain, using eGFP as a reference. We generated several lentiviral vectors encoding eGFP with different N-terminally fused small epitope tags (AU1, flag, 3flag, HA, myc and V5). After confirmation of their functionality in cell culture, we injected these lentiviral vectors stereotactically into the striatum of rats and prepared paraformaldehyde fixed floating sections for immunohistochemical analysis. Using multiple antibodies and antibody dilutions per epitope tag, we extensively assessed the efficiency of several anti-tag antibodies for chromogenic immunohistochemical detection of the epitope tagged eGFPs by determining the proportion of immunoreactivity detected by anti-tag antibodies compared to anti-GFP antibody. Using fluorescence immunohistochemistry and confocal microscopy, we also quantified the proportion of eGFP-positive cells detected by anti-tag antibodies. Our results show that all the examined small epitope tags could be detected by anti-tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. Using the presented protocol, V5/anti-V5 and HA/HA11 tag/antibody combinations provided the most sensitive detection in brain tissue. We confirmed the applicability of these optimized in vivo tag detection conditions for a difficult to detect protein, firefly luciferase (fLuc), using lentiviral vector constructs expressing V5 tagged and 3flag tagged fLuc protein.ConclusionsWe show here that several small epitope tags are useful for immunohistochemical detection of exogenous proteins in vivo. Our study also provides a generic methodology which is broadly applicable for the detection of overexpressed transgenes in mammalian brain tissue.

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“…The choice of Protein G or A beads depends on the affinity of the beaded support with the isotype of the antibody being used. For further information refer to Zhang and Chen (2001) and Bonifacino et al (2016). 12.…”

Section: Mnase Digestionmentioning

confidence: 99%

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins

Khan

Cheung

2020

Front. Cell Dev. Biol.

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The nucleosome is the principal structural unit of chromatin. Although many studies focus on individual histone post-translational modifications (PTMs) in isolation, it is important to recognize that multiple histone PTMs can function together or crossregulate one another within the nucleosome context. In addition, different modifications or histone-binding surfaces can synergize to stabilize the binding of nuclear factors to nucleosomes. To facilitate these types of studies, we present here a step-bystep protocol for isolating high yields of mononucleosomes for biochemical analyses. Furthermore, we discuss differences and variations of the basic protocol used in different publications and characterize the relative abundance of selected histone PTMs and chromatin-binding proteins in the different chromatin fractions obtained by this method.

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Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging

Cited by 4 publications

References 15 publications

Detection of Protein‐Protein Interactions by Coprecipitation

Detection of Protein‐Protein Interactions by Coprecipitation

Immunohistochemical detection of transgene expression in the brain using small epitope tags

The Use of Mononucleosome Immunoprecipitation for Analysis of Combinatorial Histone Post-translational Modifications and Purification of Nucleosome-Interacting Proteins

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