Biology Reference
In-Depth Information
SDS-PAGE) are pooled and concentrated at 4
C to 2 ml with Centriprep 30 K
(Amicon).
3. Size Exclusion Chromatography
: Next, the sample is loaded onto a refrigerated
home-packed Sephacryl S300 HR column (1.6
60 cm) that has been
preequilibrated in buffer C (20 mM Tris, 1 mM EDTA, 1 mM sodium azide, pH
7.5) at 4
C. Proteins are eluted at 1 mL/min, due to column pressure requirements
and to allow maximum peak resolution, collecting 4 mL fractions. The main
fractions containing BtubA and/or BtubB are then pooled and concentrated to
about 0.5 mL at 4
C with Centriprep 30 K. Note that BtubA and BtubB partially
separate during the two chromatographic steps (2 and 3) and that the fractions
should be chosen in this step to approach a 1:1 ratio of BtubA to BtubB. At this
stage, purified BtubA/B is partially active in quantitative assembly experiments.
4. Assembly
/
Disassembly Cycle
: To induce BtubA/B polymerization, 1 mMEGTA,
5 mM MgCl
2
, 2 mM GTP, and 300 mM potassium glutamate (from a 3 M stock
solution) are added to BtubA/B (
4 mg/mL final concentration; see
measurement below). All components are mixed well avoiding foam formation
and incubated for 10 min at 25
C without agitation. The mixture is then
centrifuged for 30 min at 100,000
g
25
C (in a prewarmed rotor). The
semitransparent assembled protein pellet (P1) (about 40% of the purified
BtubA/B) can be easily distinguished. After the supernatant is separated, the pellet
is carefully redissolved in one volume of cold 20 mMTris/HCl, 1 mMEGTA, pH
7.5, buffer, avoiding foam formation, during 20 min on ice to allow for the
complete depolymerization of the BtubAB-assembled polymers. The solution
is then centrifuged another 30 min, at 100,000
g
(at 4
C in a second, precooled
rotor). BtubA and BtubB are now recovered in the supernatant (S2), which must
be clear and colorless, indicative of no protein aggregation or assembly. Finally,
the protein is concentrated to approximately 0.5 mL and stored at
75
C.
5. Protein Concentration Measurement
: The BtubA/B concentration is accurately
measured following a spectrophotometric method which takes into account the
contribution of the bound guanine nucleotide. First, we determine the nucleotide
concentration after extraction from small protein aliquots, diluted 25-50 times
into ice-cold 0.5 NHClO
4
(final concentration). The acid solution is incubated on
ice for 10 min and the denatured protein is removed by centrifugation (10 min,
10,000
g
,4
C). The guanine nucleotide remains in the supernatant and its
concentration is then measured employing an extinction coefficient
12,400 M
1
cm
1
at 254 nm (
Correia, Baty, & Williams, 1987
). Second, the
BtubA/B concentration is measured spectrophotometrically from its absorption
spectrum in 6 M guanidinium chloride, after subtraction of the absorption due to
the guanine nucleotide, employing a protein extinction coefficient
86,550 M
1
cm
1
at 280 nm (calculated from the protein sequence) and a
nucleotide extinction coefficient 8100 M
1
cm
1
at 280 nm (
Diaz et al.,
2001
). Note that these BtubA/B preparations could also be approximately
measured employing a practical value of extinction coefficient of
100,000
3000 M
1
cm
1
at 280 nm in aqueous buffer, including the bound
