کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10825946 1064688 2011 16 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis
چکیده انگلیسی
Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation).
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 53, Issue 4, April 2011, Pages 437-452
نویسندگان
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