Direct chemiluminescent immunodetection of proteins in agarose gels.
Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.
Smejkal
Gary B
GB
Cleveland State University, Department of Chemistry, Cleveland, OH 44115, USA. gary.smejkla@proteomesystems.com
Shainoff
John R
JR
Yakubenko
Anna V
AV
D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000737:chemistry; D008163:Luminescent Measurements; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry*
D011506:Proteins; D012685:Sepharose; D006735:Horseradish Peroxidase