Linked Life Data

3127468

Source:http://linkedlifedata.com/resource/pubmed/id/3127468

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
1988-5-11
pubmed:abstractText
We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0022-1759
pubmed:author
pubmed:issnType
Print
pubmed:day
6
pubmed:volume
108
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
115-22
pubmed:dateRevised
2011-11-17
pubmed:meshHeading
pubmed:year
1988
pubmed:articleTitle
Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.
pubmed:affiliation
Department of Immunology, St. George's Hospital Medical School, London, U.K.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't