Preparation of Yeast Nuclear Envelope, Nuclear Pore Complex, Nuclei, and Spindle Pole Body
- Carbonate Extraction of Nuclear Envelopes
Purpose: to extract proteins from the nuclear envelope using carbonate
- Heparin Extraction of Nuclear Envelopes
Purpose: to extract proteins from the nuclear envelope using heparin
- Rapid, Small-Scale Subcellular Fractionation of Yeast to Produce Nuclei and Nuclear Envelopes
Yeast nuclei and NEs are prepared on a small scale using the methods below, which are modifications of previously described protocols, where detailed solution formulations can be found (Rout and Kilmartin, 1990; Rout and Kilmartin, 1994; Strambio-de-Castillia et al., 1995).
- Isolation of Yeast Nuclear Pore Complexes and Nuclear Envelopes
Here we describe two isolation procedures for yeast, one producing nuclear pore complexes (NPCs), the other nuclear envelopes (NEs) and nuclear membranes, starting from highly enriched yeast nuclei. Both result in material of sufficiently high yield and degree of enrichment to be potentially useful in a variety of preparative and analytical studies, including the identification of NPC components and their localization in the NE.
- Preparation of Yeast Nuclei and Spindle Pole Bodies
Spindle pole bodies (SPBs) are the sole microtubule organizing centers of budding yeast cells. SPBs are embedded in the nuclear envelope which remains intact during mitosis thus spindles are intranuclear. SPBs can be enriched six hundred fold and in high yield from Saccharomyces uvarum (Rout and Kilmartin, 1990). The procedure involves preparation of nuclei by a modification of an existing method (Rozijn and Tonino, 1964). The nuclei are then lysed and extracted to free the SPBs from the nuclear envelope, followed by two gradient steps to separate the SPBs from other nuclear components. These SPBs, which are about 10% pure, have been used to prepare mAbs and thereby identify components of the SPB and spindle (Rout and Kilmartin, 1990, 1991).
Immunoaffinity Purification of Protein Complexes
- Harvesting Cells and Making Noodles
This protocol is designed to harvest a yeast cell culture and prepare it for grinding.
- Cryogenic Disruption of Yeast Cells (Retsch PM 100)
Lysing of frozen yeast cells using a planetary ball mill.
- Cryogenic Disruption of Mammalian Cells (Retsch PM 100) Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels
- Conjugation of Dynabeads with Rabbit IgG
Coupling of Dynabeads with Rabbit IgG in order to produce magnetic beads which are capable of pulling out various Protein A tagged complexes.
Characterization of Proteins and Protein Complexes
- Protease Mapping
This protocol is demonstrated schematically. Tagged proteins are attached to a resin, and a partial protease digest is done. Unbound fragments are washed away, and the bound fragments are eluted and run on a gel. This method has many applications, including domain mapping (as in Devos et al, PLoS 2004). The following protocol assumes that you’re starting with already-purified protein. You can also start from yeast lysate, in which case pick up this protocol from step 7 after you’ve done your pullout, bound your protein to the beads and washed them.
General Protein Biochemistry Techniques
- Carbonate Silver Stain of Gels
This is a protocol to silver stain SDS page gels without a kit.
- Electropurification of Proteins
This protocol is for electrically eluting pure proteins from SDS-page gels. This is useful for unstructured proteins that are highly protease sensitive.
- Methanol Chloroform Precipitation of Proteins
This is a method to precipitate proteins that are not adequately precipitated by TCA/NaDOC.
- Strip Blots
The strip blot is a way to probe the same antigen with multiple antibodies at once.
- Whole Cell Lysis and TCA Precipitation
This is the method for whole cell lysis and TCA precipitation, used by the Rout lab as of 1998-2005. It is especially effective for samples with a large proportion of protein, so alternate protocols should be used if there is only a small amount of total protein in the original sample. This can be used for yeast or bacterial cells.
- Microfluidizer Protocol
The Rout lab uses a Microfluidizer® (Microfluidics, product # M11 OS) to lyse large quantities of bacteria while keeping them cool. The cells are resuspended in buffer, pushed through a patented chamber at high pressure, and passed through a cooling coil.