Biology Reference
In-Depth Information
15.4.3.2
Prepare samples of the SpAAC for time-series analysis
Perform the reaction under the same condition as described in
Section 15.4.1
. The
starting mixture consists of 100
L resin and 200
L reaction buffer (a total
m
m
L).
At different time points, take out 30
volume of 300
m
L of the resin/buffer mixture and add it into
0.4 mL of precooled reaction/wash buffer in a clean 1.5 mL Eppendorf tube
to quench the reaction. Centrifuge the resin to remove the labeling reagents.
Wash the resin once with 0.4 mL of reaction/wash buffer (30 min).
Elute the labeled rhodopsin using the elution buffer containing the C9 peptide
(15
m
m
L elution buffer
2).
15.4.3.3
In-gel fluorescence
The in-gel fluorescence experiment is carried out in the same way as described in
Section 15.4.2.3
. Lacking the knowledge for the exact concentrations of time-series
samples, we load 10
L eluate to the SDS-PAGE gel for each time point. Wash the
gel briefly before scanning it on a Typhoon 9400 fluorescence scanner.
m
15.4.3.4
Silver staining
After acquiring the in-gel fluorescence image, immediately soak the SDS-PAGE
gel with the fixation buffer for at least 1 h with two or three buffer changes
or overnight to fix the gel.
Wash the gel thoroughly washed with ultrapure water for over 1 h with at least
three changes to remove all the acetic acid and methanol.
Meanwhile, prepare the staining solution. Prepare Solution A (dissolve 0.4 g
silver nitrate in 2 mL of ultrapure water), and Solution B (add 0.7 mL of 30%
ammonium hydroxide to 10.5 mL of 0.36% sodium hydroxide solution).
Both solutions should be prepared fresh. Add Solution A dropwise into Solution
B under stirring to make the staining solution. The brown precipitate should
clear in several seconds. Add 37 mL ultrapure water to the silver-ammonia
complex solution to bring the total volume to 50 mL.
Transfer the gel into a clean container, and stain with the staining solution under
gentle agitation.
After 15 min, rinse the gel with ultrapure water to remove the residue staining
solution. Then remove the water and add freshly prepared developing buffer
(mix 0.5 mL of 1% citric acid with 50
L 38% formaldehyde, then add into 100 mL
water) to visualize the band. Collect the used staining solution in a waste bottle.
Monitor closely the development process to avoid overstaining. When the
background begins to turn yellowish, stop the reaction by rinsing the gel with 1%
acetic acid.
Scan the gel on a regular image scanner to acquire the digital data.
m
15.4.3.5
Data analysis
Analyze the signal intensity of in-gel fluorescence and silver staining data using
ImageJ. As rhodopsin runs as a smear on the SDS-PAGE gel, the entire
region of the smear should be included when measuring the overall signal
