The adsorption of large proteins in electrofocusing on immobilized pH gradients: II. Dependence on the oligomeric state of Immobiline.
Five proteins with molecular mass in excess of 200 kDa were found to adsorb onto gels during isoelectric focusing on immobilized pH gradients (IPGEF). To probe for the mechanism of that adsorption, the homogeneity of the six Immobiline preparations used to make IPGEF gels was tested. Five of these Immobiline preparations appear homogeneous in gel filtration of Sephadex G-10. The sixth Immobiline (pK 9.3) exhibits a minor component eluting ahead of the major peak and comprising less than 4% of the total Immobiline absorbing at 226 nm. The proportion of the minor component increases with column load. Major and minor components when isolated appear to equilibrate with one another. Judging by the results of mass spectrometry, all 6 preparations are free of small aggregates of less than 500-600 Da molecular mass. Ultrafiltration of the Immobiline preparations through a membrane with 500 Da nominal cutoff leads to partial desorption of only 3 of the 5 adsorbed proteins. CHAPS is ineffectual in desorbing the 5 proteins from the IPG gel made with ultrafiltered Immobilines. None of the 6 Immobiline preparations used precipitates ferritin. All large proteins that adsorb onto IPGEF gels in the pH range 4-9.5 also adsorb onto commercial IPGEF gels in the pH range 4-7.
Fawcett
J S
JS
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, Bethesda, MD 20892.
Vicchio
D
D
Chrambach
A
A
D000178:Acrylamides; D000327:Adsorption; D004798:Enzymes / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification*
D000178:Acrylamides; D004798:Enzymes; D011506:Proteins; C059413:Immobiline