N-Terminal protein sample preparation:
Proteins/Peptides in solution: For best results an optimal amount to load on the sequencer is 100 pmole of peptide or protein. Samples should be as homogeneous as possible and free of salts, detergents, lipids and other non-volatile ingredients (SDS, TRIS, etc.). Solutions used in dissolving samples should, therefore, contain only volatile components such as water, acetonitrile, etc. and be concentrated in a volume of no more than 60 ml. If your sample is supplied dried, it will be brought up in a 1:1:1 mixture of acetonitrile, water and acetic acid unless otherwise specified.
Sample Purity -- What to Avoid:
Sample purity is one of the most critical factors for successful protein sequencing.
Cut 8 pieces of Watmann paper in size 14 x 16 cm. Equilibrate PVDF membrane (smooth side up) in 10 mM CAPS, pH 11.0 buffer for ~ 10 min and then for ~ 5 min in 10 mM CAPS, pH 11.0/10% methanol. 4 Watmann paper soaked by 10 mM CAPS buffer place on semidry apparatus, remove bubbles by rolling with glass rod (further remove bubbles at each step). PVDF membrane put over the paper. Soak the gel in CAPS/10% methanol solution and place over the membrane. Remaining 4 pieces of paper wet in the same solution put over the gel. Close the apparatus, put ~ 2 kg load and run: 12V for 2 h. Before staining wash membrane 3 times with H2O.
for 0.5-1 min in 0.04% Coomassie brilliant blue R-250 prepared in 50% methanol/10% acetic acid.
rinse the membrane for 1 - 2 min with 50% methanol/10% acetic acid For proteins/peptides adsorbed onto PVDF, again the optimal amount protein/peptide to load on the sequencer is 200 pmole. The piece of PVDF should be no larger than 3 x 7 mm and the number of pieces no more than five. Keep in mind that transfer is not 100% efficient so load an adequate amount of protein or peptide onto the gel prior to electrophoresis. Remember that the intensity of the stain is not indicative of sample amount and that the presence of a single band does not always indicate that there is only one species present.
Please, note that nitrocellulose cannot be used in our Protein sequencer