When treating such acidic rCIα1 solutions under high temperature as we normally do before loading protein gels, we were unable to detect them in Western blot, suggesting that the combination of acidic buffer and high temperature could be detrimental to collagen integrity.
The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips.
Consequently, extra purification steps are required to remove these secondary metabolites from the feedstream before loading to Protein A resin [ 17].
Before loading onto the gel, the proteins were denatured by addition of a reducing agent (which included dithiothreitol, DTT) and heating to 70°C for 10 min. Each gel was run at 200 V constant for 35 min. After the electrophoresis, the gel was fixed with acetic acid/methanol solution, stained with colloidal Coomassie blue overnight, and destained with deionized, distilled water for 7 h.
The pH of the serum was adjusted to 8.0 by mixing with /10th volume 1 M Tris HCl buffer, pH 8 before loading on the Protein A-Sepharose.
The protein sample was rebuffered via dialysis into salt-free buffer and filtered before loading onto the pre-equilibrated column (50 mM MES, pH 6.5).
Next, we tested GTP exchange by stripping endogenous nucleotide, then pre-loading the Rab7 proteins with GDP before incubation with H-GTP.
For western blotting, aliquots of total protein preparations or membrane protein preparations was mixed well with an equal volume of 2× SDS loading buffer containing 100 mM DTT, maintained at room temperature for 30 min before loading onto 8% Precise Protein Gels (Pierce , Life Technologies Grand Island, NY, United States).
Unlike for samples of soluble proteins, which need be boiled before loading, membrane protein samples cannot be boiled since boiling results in further aggregation.
