First, the molecular weight of the protein sample was established to determine the state of the MBP within the monolayer by applying the equation for gases to the so-called gaseous zone of the monolayer for the conditions under which the expression holds.
While the altered molecular weight of the protein sample shown in Figure 6, lane 9 may be attributed to proteolysis, most of the protein samples in Figure 6 displayed a similar estimated molecular weight of 55 kDa.
Protein molecular weight marker standards (GE healthcare) were used for column calibration and generation of standard curve to identify the molecular weights of the test protein sample.
The molecular weight and the second virial coefficient of a protein sample in a solution can be measured using the static light scattering (SLS) technique.
Since the contrast in cryo-sample images is from the scattering differences between the ice, protein, and lipid/detergent, the protein sample may be invisible if the molecular weight of the protein is low [ 48].
Protein samples (20 μl) were concentrated on 6 % SDS-PAGE concentrated gels and separated on 10%% (for macromolecular protein) or 15%% (for small molecular protein) SDS-PAGE separation gels and blotted on a polyvinylidene fluoride membrane (Merck Millipore, Germany).
Benchmark prestained protein ladder (Invitrogen) was added to one well as a control to monitor the molecular weight of the protein samples.
To remove high molecular weight aggregate, the protein samples were centrifuged at 15,000 rpm for 30 min prior to each experiment, and the upper portion of the supernatant (the concentration was measured again at UV 280 nm) was pipetted and loaded into a quartz cuvette.
To resolve high-molecular-mass proteins, samples were run on 3 8% Tris-acetate NuPage gels.
Protein samples and molecular weight markers (Precision Protein Standards, BioRad, Milan, Italy) were separated on a 12% SDS-PAGE in reducing conditions and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA).
