Journal
/
Biologia
/
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
JoVE Journal
Biologia
É necessária uma assinatura da JoVE para visualizar este conteúdo.  Faça login ou comece sua avaliação gratuita.
JoVE Journal Biologia
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

DOI:
10.3791/54389-v

•

10:36 min

•

July 27, 2016

•

,
1Department of Biochemistry,Brandeis University, 2Winship Cancer Institute,Emory University School of Medicine

Capítulos

  • 00:05Título
  • 00:38Harvesting S. cerevisiae Cells
  • 01:44Lyse Cells and Lysate Clarification
  • 04:43Resin Equilibration and Binding and On Column TEV Cleavage
  • 06:47Packing and Eluting from Calmodulin Resin
  • 08:20Results: TAP Purification of U1 snRNP Complex in S. cerevisiae
  • 09:55Conclusion

Summary

Tadução automática

Tadução automática

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Tags

Keywords: Native Macromolecular ComplexPurificationTandem Affinity TagStructural StudyS. CerevisiaeCell LysisLiquid NitrogenCoffee GrinderCentrifugationBuffer ConditionsProtein Complex

Article

Embed

ADICIONAR À PLAYLIST

Usage Statistics

Vídeos Relacionados

check_url/pt/54389?article_type=v

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes
check_url/pt/54389?article_type=v

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting
check_url/pt/54389?article_type=v

Analyzing Large Protein Complexes by Structural Mass Spectrometry
check_url/pt/54389?article_type=v

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
check_url/pt/54389?article_type=v

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
check_url/pt/54389?article_type=v

Identification of Protein Interacting Partners Using Tandem Affinity Purification
check_url/pt/54389?article_type=v

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
check_url/pt/54389?article_type=v

High-throughput Purification of Affinity-tagged Recombinant Proteins
check_url/pt/54389?article_type=v

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
check_url/pt/54389?article_type=v

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Read Article