Diagenode

Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.


Hofemeister H, Ciotta G, Fu J, Seibert PM, Schulz A, Maresca M, Sarov M, Anastassiadis K, Stewart AF

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).

Tags
Bioruptor
Chromatin Shearing
ChIP-qPCR
Cell Lysis
Western Blot
Antibody

Share this article

Published
April, 2011

Source

Products used in this publication

  • Antibody ChIP icon
    C15200054
    Ty1 monoclonal antibody

Events

  • ASHG
    Houston, TX
    Oct 15-Oct 19, 2019
  • ddd
    dd
    Oct 18-Oct 26, 2019
  • Neuroscience 2019
    Chicago, IL
    Oct 19-Oct 23, 2019
 See all events

       Site map   |   Contact us   |   Conditions of sales   |   Conditions of purchase   |   Privacy policy   |   Diagenode Diagnostics