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.
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.