Protein Protocols Protein Protocols

78 Judd
4. Run the gel as described above.
5. Blot the peptides to a PVDF membrane as described above, but add 1–2 mg of dithiothreitol
to the blot transfer buffer.
6. Fix and stain as above, again adding 1–2 mg of dithiothreitol to the fixer/destainer, CBB,
and dH 2O used to rinse the peptide-containing PVDF membrane.
4. Notes
1. Working range of separation about 40 kDa down to about 1 kDa.
2. Working range of separation about 20 kDa down to less than 500 Dalton.
3. Use electrophoresis grade SDS. If peptide bands remain diffuse, try SDS from BDH
(Poole, Dorset, UK).
4. Degassing of gel reagents is not necessary.
5. Coomassie staining can generally visualize a band of 0.5 µg. This may vary considerably
based on the properties of the particular peptide (some peptides stain poorly with Coomassie).
Some peptides do not bind SDS well and may never migrate exactly right when
compared to mol-wt markers. Fortunately, these situations are rare.
6. Protocols are designed for a standard 13 cm × 11 cm × 1.5 mm slab gel. Dimensions and
reagent volumes can be proportionally adjusted to accommodate other gel dimensions.
7. Permanent marks with a diamond pencil can be made on the back of the back plate if the
plate is dedicated to this gel system.
8. The depth of the spacer gel can be varied from 1 to 2 cm. Trial and error is the only way to
determine the appropriate dimension for each system.
9. It is wise to feel the front plate several times during the electrophoresis to check for overheating.
The plate will become pleasantly warm as the run progresses. If it becomes too
warm, the plates might break, so turn down the power!
10. Standard-sized gels can be fixed in as little as 4 h with shaking.
11. It is best to blot peptides to PVDF membranes rather than nitrocellulose membranes, since
small peptides tend to pass through nitrocellulose without binding. Moreover, peptides
immobilized on PVDF membranes can be directly sequenced in automated instrumentation
equipped with a “blot cartridge” (6).
12. The pH of the transfer buffer can be varied from 5.7 to 8.0 if transfer is inefficient at
pH 8.0 (7).
13. Wear disposable gloves when handling membranes.
14. Do not refrigerate the gel. It will contract and pull away from the plates, resulting in leaks
and poor resolution.
15. Do not pour the stacking gel the day before electrophoresis. It will shrink, allowing the
samples to leak from the wells.
Acknowledgments
The author thanks Joan Strange for her assistance in developing this system, Pam
Gannon for her assistance, and the Public Health Service, NIH, NIAID (grants RO1
AI21236) and UM Research Grant Program for their continued support.
References
1. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680–695.
2. Judd, R. C. (1988) Purification of outer membrane proteins of the Gram negative bacterium
Neisseria gonorrhoeae. Analyt. Biochem. 173, 307–316.