protein size determination? - (Feb/26/2008 )
How can you determine the exact size (kDa) of a native protein, possibly a dimer?
You can always calculate it according to the amino acid sequence. The average weight of an amino acid is 110Da but you would need to look up each amino acid and do the math if you need exact. Otherwise, you can run a native gel (if you want to see the dimer) but proteins can always migrate a bit different than the predicted size in a gel.
in addition to rkay47:
if you have it pure or at least enriched, run gel filtration or a sucrose gradient in ultracentrifugation; precise calibration is obligatory
How accurate can you estimate protein size from native gel? What means blue native gel compare to regular native gel?
it is reliable, however, to find the right running conditions, particularly the right buffer system, is tedious work;
I do not know the difference between blue and regulare native gel; go ask mdfenko
Easiest way is to run a SDS-PAGE. If you run a PAGE it won't give you any useful information about size of your protein. In SDS-PAGE proteins are denatured and are given a negative charge proportional to their mass.
I do not agree that PAGE give any useful information about size since in pI-appropriate native PAGE protein runs in retention to its size
I do not agree that PAGE give any useful information about size since in pI-appropriate native PAGE protein runs in retention to its size
What you mean? I didn't say PAGE, you said PAGE
it is reliable, however, to find the right running conditions, particularly the right buffer system, is tedious work;
I do not know the difference between blue and regulare native gel; go ask mdfenko
I said SDS-PAGE!
It is a standard method for determining size, so standard that you can find it in almost every biochemistry text book.
As I said PAGE won't give you any information about size. If you set pH to pI how do you suppose the protein is going to move in electric field? and decide do you want to set pH to pI of unknown protein or your marker?
take your words back or show me a reference.
Cheers,
DG
With a properly calibrated column, gel filtration will give you a good size of the dimer, after an SDS-PAGE gives you the monomer size. For a more precise number, look at MALLS (Multi angle laser light scattering) which can be added to a standard HPLC system to give very good size data. If you want data on the interaction, analytical ultracentrifugation is the way to go..
The purpose is to see if the protein is dimer. So I cannot use SDS-PAGE because it will denaturate the protein. Unfortunately I dont have access to gel filtration HPLC. So I have to use PAGE. Can blue native PAGE (BN-PAGE) solve this problem? I heard that in BN-PAGE the proteins will migrate according to their molecular size but still stay in native form.