Table 1.

Typical settings for preparing various macromolecular complexes using a Vitrobot™

	Intermediate Filaments	Kinesin-Decorated Microtubules	Membrane Proteins/Liposomes
Cryogen	Ethane/propane mix	Ethane	Ethane/propane mix
Chamber Temperature	22°C	22°C	22°C
Chamber Humidity	85%	90%	70%
Blot Total	1	1, 1	1
Blot Force	0	0, 0	1
Blot Time (s)	2	1.5, 3	3
Drain Time (s)	0	0, 0	0
Wait Time (s)	3-10	35, 60	Manual
Special Considerations	•Apply 2 μl of sample to each side of grid	•Apply 5 μl of microtubules first, blot using 1st set of values listed. Then add 5 μl of motors and blot using 2nd set of values listed.	•Apply 2 μl of sample to each side of grid.
			•No glow discharge prior to sample application.