Protocols

Below is a working list of NCDIR protocols and methodologies. Please contact us if you have questions, comments and/or requests.

Disruption of Yeast or Bacteria Cells

• Harvesting Cells and Making Noodles
• Harvesting Cells and Making Noodles (video protocol)
• Cryogenic Disruption of Yeast Cells PM100
• Cryogenic Disruption Using PM100 Ball Mill (video protocol)
• Microfluidizer
• Zymolase Treatment

Disruption of Mammalian Cells

• Protein Affinity Capture from Cryogenically Disrupted Human Cells  (video protocol)
  
• Protein Affinity Capture from Cryogenically Disrupted Human Cells (pdf)

Protein Biochemistry

• Carbonate Silver Stain
• Electropurification
• Methanol Precipitation of Protein
• Protease Mapping
• Strip Blots
• Whole Cell Lysis and TCA (Trichloroacetic acid) Precipitation

Subcellular Fractionation

• Isolation of Yeast Nuclear Envelopes and Nuclear Pore Complex 
• Preparation of Yeast Nuclei & Spindle Pole Bodies 
• Rapid, Small-Scale Subcellular Fractionation for Nuclei and Nuclear Envelopes
• Heparin Extraction of Nuclear Envelopes  
• A general method for quantitative fractionation of mammalian cells

RNA Exosome Protocols

• Affinity Proteomic Analysis of the Human Exosome and Its Cofactor Complexes
• RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells
• Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
• Purification and analysis of endogenous human RNA exosome complexes

Isolation of Protein Complexes

• Conjugation of Dynabeads (M270 Epoxy)
• Conjugation of Tosylactivated Dynabeads
• Protein Complex Purification by Affinity Capture
• Multi-well Interactomic Screen Affinity proteomics to study endogenous protein complexes
• Native Elution of Yeast Protein Complexes Obtained by Affinity Capture
• Density Gradient Ultracentrifugation to Isolate Endogenous Protein Complexes after Affinity Capture
• Optimized Affinity Capture of Yeast Protein Complexes
• Affinity Purification of the RNA Degradation Complex, the Exosome
• Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization
• Improved Native Isolation of Endogenous Protein A-Tagged Protein Complexes
• Affinity Proteomics to Study Endogenous Protein Complexes
• Characterization of L1-Ribonucleoprotein Particles
• Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization
• High-Efficiency Isolation of Nuclear Envelope Protein Complexes from Trypanosomes 
• High-Yield Isolation and Subcellular Proteomic Characterization of Nuclear and Subnuclear Structures from Trypanosomes
• Affinity Isolation of Endogenous Saccharomyces Cerevisiae Nuclear Pore Complexes
• Affinity Purification of Protein Complexes (including high pH elution steps 12 & 13)
• Conjugation of Magnetic Beads for Immunopurification of Protein Complexes

Mass Spectrometry

• MALDI Sample Preparation_In-gel Digestion and Crystallization
• MALDI Sample Preparation_The Ultra Thin Layer Method MALDI
• Sample Preparation_Saturated 4-HCCA Solution
• Native mass spectrometry analysis of affinity-captured endogenous yeast RNA exosome complexes

Other Protocols

• ODELAM: Rapid sequence-independent detection of drug resistance in isolates of Mycobacterium tuberculosis.
• Measuring in vivo protein turnover and exchange in yeast macromolecular assemblies 
• Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements