Protein Ladder Separations
We’ve recently put together our own protein ladder, which contains myosin heavy chain, beta-galactosidase, phosphorylase b, IgG, albumin, and cytochrome c. This is my first attempt running this ladder on w398, one of the new pinhole free membranes. From the TEM pics, it seems that w398 has pores all around the 50nm range, which is a bit too big for protein separations.
The gel shows us that myosin and phosphorylase b do not pass into the filtrate. It is a little surprising that phos b doesn’t go through while beta-gal does. Denatured, phos b is 95kD, but should naturally be a dimer at 190kD. Beta-gal runs at 116kD, but naturally should be a multimer at 464kD. It’s possible that in our sample phos b is a dimer and beta-gal a monomer.
It’s possible that the phos b was denatured, as the solution seemed more viscous than I remember. I am surprised though that a denatured protein (and presumably just a long chain) wouldn’t go through. Although this sample may now be made up of protein aggregates.
Jess –
What causes the proteins to denature? In my simplified understanding, if the protein denatures and unfolds there is an increased chance of tangling, right? Is this what happened to the phos b?
I’m also still confused about the units on the right (kD) and numbers on the left. Why don’t they match? You explained this to me a while ago, but I’ve forgotten now…
Can the Malvern tell you anything about your protein solutions? It seems like monomer/dimer differences should be visible?
Do any any of these proteins interact with each other? Do they have similar pKa?
I doubt an extremely long denatured protein could ever diffuse through the membrane, unless it formed a ball. Under flow conditions, maybe, as long as it did not clump with other proteins…
Denatured proteins typically make huge aggregates. If you are right about PhosphoB, then the results make sense and this is worth repeating with fresh protein. The Malvern should definitely reveal the difference between fresh (natured) and denatured protein.