Change the pump speed to 100 μL/min and restart the pump.
Allow the running buffer to flush through the system to ensure
removal of free linker (approximately 10 min).
14. Prepare 10 μg/mL avidin/streptavidin solution. Dilute 10 μL
of 1 mg/mL avidin/streptavidin stock with 990 μL of running
buffer in a 1.7-mL tube and mix by pipetting.
15. Temporarily stop the pump. Move the inlet tube to the avidin/
streptavidin solution in the 1.7-mL tube. Ensure that no air
bubbles are present within the tubing. Change the pump speed
to 50 μL/min and restart the pump. Wait for f and D signal to
plateau to ensure saturation of surface (see Note 22).
16. Temporarily stop the pump. Move the inlet tube back into the
running buffer ensuring that no bubbles are present in the
tubing. Change the pump speed to 100 μL/min and restart
the pump. Allow the running buffer to flush through the
system to ensure removal of free avidin/streptavidin (approximately 15 min).
17. Surface is now functionalized and ready for the immobilization
of biotinylated molecule and further interaction analysis.
Repeat step 16 with molecules of interest at appropriate
concentrations.
4 Notes
1. If CaCl 2 used to prepare buffer is nonanhydrous/partially
hydrated, ensure that the added molecular weight of water
content is included in calculating mass needed for 100 mM
solution. CaCl 2 is not always required for lipid bilayer production using zwitterionic lipids; however, we have found that
using CaCl 2 produces consistency in results with varied lipid
mixtures.
2. Buffers can be degassed in three ways. Vacuum: place buffer in a
flask with a side arm attached to a vacuum pump. Add a stir bar
to the flask and seal the flask at the top using a rubber stopper.
Place the flask on a stir plate, and under low vacuum, stir buffer
at medium speed for a minimum of 1 h. Vacuum and sonication: it is the same as the previous one; however, the stir
bar/plate can be omitted and the flask under low vacuum can
be placed in a sonicator water bath. Sonicate for approximately
1 h. Helium sparging: place a sparging frit to the end of a
helium line and place into the buffer. Turn on the helium at a
very low pressure for approximately 5 min. Always ensure that
filtering is performed prior to degassing as the filtration process
will aerate your solution.
192
Holly L. Birchenough and Thomas A. Jowitt
Allow the running buffer to flush through the system to ensure
removal of free linker (approximately 10 min).
14. Prepare 10 μg/mL avidin/streptavidin solution. Dilute 10 μL
of 1 mg/mL avidin/streptavidin stock with 990 μL of running
buffer in a 1.7-mL tube and mix by pipetting.
15. Temporarily stop the pump. Move the inlet tube to the avidin/
streptavidin solution in the 1.7-mL tube. Ensure that no air
bubbles are present within the tubing. Change the pump speed
to 50 μL/min and restart the pump. Wait for f and D signal to
plateau to ensure saturation of surface (see Note 22).
16. Temporarily stop the pump. Move the inlet tube back into the
running buffer ensuring that no bubbles are present in the
tubing. Change the pump speed to 100 μL/min and restart
the pump. Allow the running buffer to flush through the
system to ensure removal of free avidin/streptavidin (approximately 15 min).
17. Surface is now functionalized and ready for the immobilization
of biotinylated molecule and further interaction analysis.
Repeat step 16 with molecules of interest at appropriate
concentrations.
4 Notes
1. If CaCl 2 used to prepare buffer is nonanhydrous/partially
hydrated, ensure that the added molecular weight of water
content is included in calculating mass needed for 100 mM
solution. CaCl 2 is not always required for lipid bilayer production using zwitterionic lipids; however, we have found that
using CaCl 2 produces consistency in results with varied lipid
mixtures.
2. Buffers can be degassed in three ways. Vacuum: place buffer in a
flask with a side arm attached to a vacuum pump. Add a stir bar
to the flask and seal the flask at the top using a rubber stopper.
Place the flask on a stir plate, and under low vacuum, stir buffer
at medium speed for a minimum of 1 h. Vacuum and sonication: it is the same as the previous one; however, the stir
bar/plate can be omitted and the flask under low vacuum can
be placed in a sonicator water bath. Sonicate for approximately
1 h. Helium sparging: place a sparging frit to the end of a
helium line and place into the buffer. Turn on the helium at a
very low pressure for approximately 5 min. Always ensure that
filtering is performed prior to degassing as the filtration process
will aerate your solution.
192
Holly L. Birchenough and Thomas A. Jowitt