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United States Department of Agriculture

Agricultural Research Service

Title: Inexpensive Chemifluorescent Detection of Antibody Alkaline Phosphatase Conjugates on Western Blots Using 4-Methylumbelliferyl Phosphate.

Authors
item Weiland, John
item Anderson, James
item Bigger, Brant

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2006
Publication Date: May 3, 2006
Citation: Weiland, J.J., Anderson, J.V., Bigger, B. 2006. Inexpensive chemifluorescent detection of antibody - alkaline phosphatase conjugates on Western blots using 4-methylumbelliferyl phosphate. Analytical Biochemistry. 361:140-142.

Interpretive Summary: Numerous chemicals are available for the detection of protein antibody complexes in research. Many of these substrates are expensive and require costly infrastructure for their use. The chemical 4-methylumbelliferyl phosphate (4-MUP) is an inexpensive and well-studied compound in biochemical research, yet has not found popular use in many laboratories. With the advent and inclusion in most laboratories of standard digital camera systems for the recording of images excited by UV-light, the use of 4-MUP offers an inexpensive compound to exploit this technology. It is anticipated that 4-MUP will find use in teaching, research, and clinical laboratories where such imaging stations are found.

Technical Abstract: The phosphatase substrate 4-methylumbelliferyl phosphate was tested as a fluorescent reporter for the detection of alkaline phosphatase (AP)-conjugated antibody on immunoblots. Dilution of bovine serum albumin (BSA) was used as a protein target with the use of anti-BSA antisera as the primary reactant. Detection of this complex on nitrocellulose membranes with AP-conjugated secondary antibody indicated that use of 4-MUP is similar in sensitivity to the commonly-used NBT/BCIP substrate, yet is significantly less expensive per blot performed. The substrate 4-MUP was used to demonstrate the detection of beet necrotic yellow vein virus in infected sugarbeet seedlings and of polyphenol oxidase enzyme in wild oat. Application of the substrate is anticipated where high sensitivity is not required and where cost of operation is limiting, such as in teaching laboratories.

Last Modified: 9/21/2014
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