Protein Ladder
I’ve tried to collect some information on the protein ladder that we’ve been using for separations. Sizes have thus far been taken from crystal structures and loosely measured used JMol on the PDB site. This is meant to just give us an idea of dimensions; hydrodynamic data would probably be better.
pI and charge information was found using a protein calculator from Scripps. This tool works by taking a protein sequence and pI data from free amino acids to calculate pI and charge at a specific pH. It is important to note that this is not an accurate way to find protein charge, as folding and environment makes a big difference (I have first hand knowledge of this from my undergrad research w/ Ravi Bassavappa and Ben Miller, there can be some really huge pI shifts).
| Protein | Mol Weight (monomer) | Size (nm) | pI | Charge | PDB ID |
| myosin heavy chain | 200 | 160 | 5.46 | -84 | GenBank: AAG43572.1 |
| b-galactosidase | 116 | 16 | 5.51 | -32.9 | 1dp0 |
| phosphorlase b | 97 | 8.3 | 7.21 | 2.1 | 1gpb |
| albumin | 67 | 7.2 | 5.96 | -12.2 | 1ao6 |
| ovalbumin | 45 | 6.3 | 5.29 | -10.5 | 1ova |
| carbonic anhydrase | 31 | 4.2 | 7.12 | .5 | 1v9i |
| trypsin inhibitor | 25 | 3.4 | 4.77 | -10.7 | 1avu |
| lysozyme | 14 | 3.1 | 9.04 | 7.9 | 2vb1 |
Notes:
Myosin is a very long linear molecule and may tend to behave differently than the other proteins in separations.
Molecules smaller than b-galactosidase are more similar in size than their molecular weights indicate. However, we have not had cutofs low enough to really view the precision of separations in this regime. In most cases we have shown we can cut between b-gal and myosin, phos b and b-gal, or albumin and phos-b. We should try carbonization and separations to see if we retain “sharpness” for these smaller proteins.
Most of these proteins tend to be negative by initial calculation. To get a better idea of charge interactions, we should get zeta potential data for each protein. DLS size would be very useful as well. I previously tried building our own protein ladder, but ended up thinking the extracts were more convenient and useful. Now that we have more knowledge about separations, it may be a good time to revisit a custom ladder.