Bart's Cookbook-- Choice of electrophoretic conditions for peptide mapping Electrophoresis Traditionally, 35S-methionine-labeled proteins have been mapped at pH 4.72 and 32P-labeled proteins at pH 8.90. Some people favor pH 1.9 for both. There are no rules. Use whatever pH gives a useful distribution of peptides. However, an issue to keep in mind is that it is valuable if your maps can be compared easily with those of other people who are mapping the same protein. If everyone else in the field is using one pH, it's sensible to use that pH too. pH 1.9 and 4.72 maps. The origin in these maps is in the lower left, up 2.5 or 3 cm from the bottom and in 5 cm from the left edge of the plate and the positive electrode is on the left. At these acidic pHs, most peptides are postively charged and therefore migrate toward the minus electrode. Electrophoresis is done for 27 min at 1 kV using the MBVL rigs or the CBS rig. 8.9 maps. The origin here is in the middle of the plate, up 3 cm from the bottom. These maps are sometimes run with the origin placed at the back of the rig. There has been occasional trouble with puddling of buffer in 8.9 maps. Since the origin is in the middle of the plate, it doesn't matter which side the anode is on. Again, electrophoresis is for 27 min at 1 kV on an MBVL rig. Tony likes to do the electrophoresis for 20 min. This gives less separation, but keeps the free phosphate on the plate. Note that these electrophoresis times are correct only for an MBVL-type rig. If another rig is used, it is likely that a different number of volt/minutes will have to be used. These should be determined empirically. Chromatography. We have two kinds of chromo buffer. Regular was developed by Wade Gibson for iodinated peptides. It is useful for hydrophobic peptides, such as those containing the amino acid labeled during iodination, tyrosine. The 32P or phosphochromo buffer was developed by Nancy Axelrod. It is less hydrophobic and gives better separation of non-hydrophobic peptides such as those containing phosphorylated amino acids. Additionally, a third buffer containing, among other things, isobutyric acid, is currently in vogue. You should use the buffer that gives the best separation of the peptides you are interested in. Traditionally, regular buffer is used for maps of 35S-methionine labeled proteins and phospho buffer for phosphorylated peptides. Back to the Table of contents