 |
 |
Dear Tassos, Your assumptions are right, if (1) your dn/dc is accurate, or (2) your machine is calibrated. We recently measured a protein of a similar size to yours, and when a 700 Da ligand was added to the buffer, the measured protein mass was increased accordingly. So MALS can be pretty accurate. For our dn/dc, for pure proteins, we always use 0.185 (not 0.19). For sugar groups, we assume a dn/dc of 0.14, and estimate a mass-averaged value for the glycoprotein (usually somewhere between 0.175 to 0.18). For DNA and RNA, the values will be different, again. You may also realize that by changing a simple calibration constant, you can modify your measured molar masses anyway you want. It may be time for a recalibration (it is not difficult, you can do it yourself). We tend to regularly run BSA, and see if everything is as expected with our equipment. Good luck, Engin Anastassis Perrakis wrote: > Dear all, > > The MALLS instruments on-line with an FPLC and with an RI detector, > should provide an 'absolute MW', shape independent, > and indeed in our hands they do well. Until yesterday, where a 21kD > protein pretends to be 25 kD. We did the mass spec > anyway, and its 21kD as we expected to the residue, but I am still > puzzled by that result. > > One central assumption for the MALLS formulas, is that dn/dc, the > specific refractive index increment, is constant for unmodified proteins, > made by aa with no sugars etc. Literature suggests dn/dc values for > proteins to be constant and between 0.189/0.190 is a good value, > with minimal buffer dependence for aqueous buffers with 'the usual' > salts. > > I am a rather bad physicist, but my reading tells me that dn/dc, and > thus light scattering, depends to the "laser-light induced dipole in > the molecule". Is there any reason to believe that in theory a > molecule with a very particular charge distribution (eg a small DNA > binding protein which is already a 'dipole') would have significantly > different dn/dc values? Is anyone aware of such an experiment? > Literature searches were in vain ... > > Best - > > Tassos