1 / 13

Identification, production and purification

Identification, production and purification. Identification methods Purification strategies Single gene expression and in vitro assembly Co-expression techniques. Choice of the purification strategy. - Most widely used tags: His, GST, strep, FLAG. - Use two different affinity tags.

valin
Télécharger la présentation

Identification, production and purification

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Identification, production and purification Identification methods Purification strategies Single gene expression and in vitro assembly Co-expression techniques

  2. Choice of the purification strategy - Most widely used tags: His, GST, strep, FLAG - Use two different affinity tags

  3. Tandem affinity purification Apply the cell lysate to the first affinity resin Wash unbound proteins and contaminants Elute tagged protein I and interacting partner with elution buffer I Apply the cell lysate to the second affinity resin Wash unbound proteins and contaminants Elute tagged protein II and interacting partner with elution buffer II Affinity resin I Affinity resin II

  4. His-tag

  5. Ni-NTA Ni-IDA

  6. GST-affinity

  7. Strep tag-II Elution with biotin analog: desthiobiotin

  8. CBP affinity

  9. Comparison of affinity tag technologies Lichty JJ 2005 PEP May 41(1):98-105

  10. Sf9 ou H5 Production of multi-protein complexes Co-infection His-tagged subunit Flag-tagged subunit GST-tagged subunit native subunit I 2 50 mM I 2 5mM FT Talon I 5mM IP Flag Wash MW His-p89 p80 His-tag p62 p44, p52 p34F Flag 1 litre, 300 ml dépot 20 ml

More Related