摘要:

AbstractIt has been impossible, up to now, to focus ferritin (a large,ca.450 000 Da, ironstorage protein ubiquitous in mammalian systems) in immobilized pH gradients (IPG). This was not a sieving problem, as ferritin focuses readily in a 4%T polyacrylamide (or polytrisacryl) gel in conventional carrier ampholyte isoelectric focusing. A strong interaction with the IPG matrix was suspected, but an auto‐buffered (pH 6.0) soluble IPG polymer failed to precipitate ferritin from solution, although it altered its visible spectrum, suggesting the formation of soluble complexes. By a Takeo plot, the dissociation constant (Kd)of the ferritin‐IPG matrix complex was estimated to be 0.1 mM. However, even a 100‐fold dilution of Immobilines into the IPG matrix with a concomitant addition of competing ions (up to 3 % carrier ampholytes) failed to produce good focusing patterns. An unsuspected, hitherto un‐reported, precipitating power of free Immobilines in solution on the ferritin macroion was found to be the key to the riddle. The precipitating ions are (in order of aggregation power): pK9.3>pK7.0>pK6.2. None of the acidic Immobilines (pKs 3.6, 4.4 and 4.6) and, among the basic, only the pK8.5, exhibit any precipitation power on ferritin and other complex protein mixtures. In a new IPG pH 4–5 interval, containing only the pK4.6 as buffering and the pK8.5 Immobilines as titrant ions, excellent focusing patterns of ferritin were obtained. It is hypothesized that the precipitating Immobilines act as cationic detergents, and are able to cross‐link macroions with a mixed‐type, hydrophobic‐ionic interaction at the two extremities of the molecule. Guidelines are given for handling this major problem on a short‐term basis, the longterm goal being the replacement of the ‘unfavorable’ Immobilines (pKs 9.3, 7.0 and 6.2) by new chemical structures of the desirable type (the non‐preci

ISSN:0173-0835    

DOI:10.1002/elps.1150080112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

摘要:

AbstractNitrocellulose membranes were activated by cyanogen bromide in order to improve their binding capacities. Crude extracts of a grass pollen,Dactylis glomerata, and a mite,Dermatophagoides farinae, were separated by isoelectric focusing in agarose and blotted by capillary transfer onto nitrocellulose or activated nitrocellulose. TheDermatophagoides farinaecomponents were visualized by Coomassie Brilliant Blue, India ink or Aurodye staining. TheDactylis glomerataandDermatophagoides farinaeallergens were revealed by radioimmunodetection using sera from allergic patients. A great increase of allergen binding was observed visually and by radioactive counting on activated nitrocellulose as compared with untreated nitrocellulose. New allergens which were not detectable on nitrocellulose appeared on activated nitrocellulose.

ISSN:0173-0835    

DOI:10.1002/elps.1150080113

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080114

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080115

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080116

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150080101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1987

数据来源: WILEY