Biology Reference
In-Depth Information
FIGURE 24.2
(NH
4
)
2
SO
4
-induced phase separation of purified rhodopsin. (A) Solid (NH
4
)
2
SO
4
was added
to rhodopsin purified in 25 mM NG (
n
), 50 mM NG (
), or 50 mM OG (
□
). [Rho]
pool
corresponds to the concentration of rhodopsin prior to treatment with (NH
4
)
2
SO
4
, and
[Rho]
conc
is the final concentration of rhodopsin after (NH
4
)
2
SO
4
-induced phase separation.
Modified and expanded from
Salom, Le Trong, et al. (2006)
. (B) Phase separation in solutions
containing 1% detergent in 100 mM MES, pH 6.35, after adding saturating amounts of solid
(NH
4
)
2
SO
4
and overnight incubation on ice (HG, heptyl glucoside; HTG, heptyl-thio-
glucoside; OG, octyl glucoside; OTG, octyl-thio-glucoside; OGNG, octyl glucoside neopentyl
glycol; NG, nonyl glucoside).
Add 0.25 volumes of 0.5 M MES, pH 6.35, to the pooled purified rhodopsin.
In a centrifuge tube, weigh
0.69 g of solid (NH
4
)
2
SO
4
per mL of sample. Take
into account that each gram of (NH
4
)
2
SO
4
will add
0.5 mL to the final volume;
therefore, more than one tube could be needed for the entire sample.
Add the rhodopsin sample to the centrifuge tube(s) (1.45 mL per g of (NH
4
)
2
SO
4
,
and stir with a small magnetic rod until the solution appears clear
(15-30 min). Use a glass rod to assist mixing during the first fewminutes until the
magnetic rod can stir by itself. Use a tube stand or clamp to keep centrifuge
tubes vertical on the magnetic stirrer.
Incubate the tube(s) on ice for 4-7 days to allow excess (NH
4
)
2
SO
4
to crystallize
out of solution. Shorter incubation periods will result in the growth of transparent
(NH
4
)
2
SO
4
crystals among the rhodopsin crystals, (
Fig. 24.3
) thereby complicating
