N-terminal Peptide Sequencing from SDS-PAGE Electroblots.
This is a very useful procedure for characterizing proteins identified from SDS-PAGE gels. Note that most proteins are N-terminally blocked, with an acetyl or other group, and so cannot be sequenced using the Edman degradation methodology. For this reason proteins are often cleaved with proteases, such a trypsin, chymotrypsin, V8 protease etc., and the N-terminal segments of the fragments determined. Use of BLAST or other database searching programs can then be used to rapidly identify the intact protein.
1. Use either Laemmli, Tris-tricine or 2D gels. Run as usual.
2. Assemble blotting apparatus as for Western blotting, but use as blotting Buffer 10 mM MES, pH= 6, 20% MEOH.
3. Use PVDF membranes -- remember it is essential to prewet these membranes prior to use with methanol. Membranes can be obtained from Biorad and other vendors.
4. Transfer conditions: overnight at 20 volts in cold box or 1 hr 90 volts. Use a powerful power supply, 100mA at least and preferably 500mA or more.
5. You can add 0.01% SDS to transfer buffer if you know your protein is not blotting under the normal conditions.
Staining PVDF Membranes
1. Wash PVDF membrane in distilled water several times in a pyrex dish. Handle membrane with forceps.
2. Stain in 0.02% Coomassie Brilliant blue in 40% methanol, 5% acetic acid for 20-30 seconds.
3. Destain in 40% methanol, 5% acetic acid for up to 1 min. Note membrane may not appear destained -- but background color will disappear after drying, and specific protein bands are visible.
4. Rinse membrane in distilled water, 3-5 min with at least 3 changes.
5. Air dry, place between two Whatman papers and wrap in foil. Can store at -20°C if required.
6. Identify band of interest, cut it out with a clean scalpel or razor blade, and take it to your local sequencing facility.
|©EnCor Biotechnology Inc. .||To go to EnCor Homepage Press Here|