Tassos, The most obvious answer (and possibly incorrect) is that DNA itself has a different dn/dc value and when you say that you have a DNA binding protein, chances are that some or all of it may be bound to DNA, which would change the nature of the beast (and the MW). Perhaps you can deliberately strip the DNA out (heparin?) and try again? Not being an expert (but we have someone in the lab who knows better than I do), I think I also understand that the smaller the protein, the less reliable results are. Then again, 21kD is not really small.
Mark -----Original Message----- From: Anastassis Perrakis <a.perra...@nki.nl> To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 27 Mar 2009 6:00 am Subject: [ccp4bb] off topic - static laser light scattering 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?
