Detection of Glu-Glu-tagged proteins in mammalian cell culture media by dot immunoblotting.
A dot immunoblotting technique has been developed to estimate the relative expression levels of tagged recombinant human proteins in mammalian cell culture media. Variations in sample denaturation, blocking agents and membrane composition and treatment were used to optimize the signal-to-noise ratio of the defined procedure. The method is rapid, with sensitivity extending to the low nanomolar range for a number of recombinant proteins. This technique should have general utility for antibody-based measurements of other tagged and non-tagged proteins in cell culture media or in biological fluids.
Ellsworth
J L
JL
ZymoGenetics, Inc., Seattle, WA 98102, USA. ellsworj@zgi.com
Hamacher
N
N
Bagwell
N
N
West
J W
JW
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004151:Dipeptides / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007668:Kidney / Q000166:cytology; D008322:Mammals; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D008894:Milk Proteins; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity
D017077:Culture Media, Conditioned; D004151:Dipeptides; D008565:Membrane Proteins; D008894:Milk Proteins; D011994:Recombinant Proteins; C094686:glutamyl-glutamic acid