UC Riverside Core Instrumentation Facility
Guidelines for Preparation of Protein Sequencing
Samples (Protein sequencing by Edman degradation has been discontinued)
1.The purity of the sample is also crucially important, since with contaminating proteins it is impossible to sort out the large number of possible sequences (210for a 10 amino acid peptide). An exception to this rule comes when the quantities of two or more proteins are very different; in this case, it is sometimes possible to sort out the different sequences because the amounts of PTH-amino acids in each cycle allow assignment to one sequence or another. A second possible exception comes when the investigator can predict the presence of a specific peptide; in this case the data can be examined for the presence of the predicted amino acids in each cycle. In this case, of course, one could only say that the data are “consistent” with the presence of a specific peptide.
2. Samples should
be submitted on PVDF membranes in an amount no more than that obtained from
two lanes of a one-dimensional gel (reducing the amount of membrane reduces
the background and increases the quality of the called amino acids) or in less
than 0.02 ml of a volatile solvent such as water, 5 mM ammonium bicarbonate,
0.05% trifluoroacetic acid (TFA), or 50% acetonitrile/0.05% TFA. Samples may
contain small amounts of SDS (less than the equivalent of 0.02 ml of 0.01% SDS).
Samples should not contain more than the equivalent of 10 µl of 25 mM
non-volatile salt and should be free of non-volatile primary amines. After blotting
samples onto PVDF, they should be stained with Coomassie Blue (silver stains
may not be used) and then destained with at least 4 changes of destaining solvent
followed by a final wash with ultrapure water to lower the very high concentrations
of Tris, glycine and other gel and transfer buffers that otherwise will interfere
with sequencing. Small amounts of Coomassie will not interfere with the sequencing
chemistry. After air drying, the bands or spots of interest should be individually
excised and placed in clean (rinsed out with methanol) and dry 1.5 ml Eppendorf
tubes. Samples in SDS polyacrylamide gels cannot be directly loaded into our
sequencer. The cycle charges for amino acid sequencing include HPLC identification
of the resulting PTHamino acids and summary tables of the estimated PTH-amino
acid yields. There is a minimum 6 cycle charge for samples which fail to sequence
or which contain less than 6 amino acids. We can sometimes monitor a sequencing
run during the day to halt collection of data, thereby minimizing charges for
blank cycles after usable data can no longer be obtained. For overnight runs
we set the instrument to run for the number of cycles specified by the investigator
or for 10 cycles followed by a pause; data quality can then be inspected in
the morning and then stopped or continued by agreement with the investigator.
3. Additional resources on general aspects of protein electrophoresis, staining and blotting for subsequent sequencing can be found at:
http://us.expasy.org/ch2d/protocols/
http://www.bioc.cam.ac.uk/pnac/blotguide.html