title: A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB.
abstract: Protein blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue or cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging protein blots in western blotting. Here we describe a methodology for stripping such western blots, already developed with the colorimetric substrate TMB on nitrocellulose membrane and reprobing the blot (after stripping) for the detection of a second protein through CL. The stripping procedure utilizes a stripping buffer consisting of β-mercaptoethanol, SDS, and Tris-HCl, while washing buffer consists of PBS added with 0.1 % Tween 20 and involves a series of steps facilitating accurate detection of the second protein in the stripped blot through CL.
authors.affiliation: Division of Endocrinology, Central Drug Research Institute, M.G. Road, Lucknow, 226031, Uttar Pradesh, India.
authors.forename: Parmita
authors.initials: P
authors.lastname: Kar
authors.forename: Saurabh Kumar
authors.initials: SK
authors.lastname: Agnihotri
authors.forename: Archana
authors.initials: A
authors.lastname: Sharma
authors.forename: Rekha
authors.initials: R
authors.lastname: Sachan
authors.forename: Madan Lal
authors.initials: ML
authors.lastname: Bhatt
authors.forename: Monika
authors.initials: M
authors.lastname: Sachdev
mesh_terms: D001560:Benzidines / Q000737:chemistry*; D015153:Blotting, Western / Q000379:methods*; D003101:Collodion / Q000737:chemistry; D003124:Colorimetry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D011506:Proteins / Q000032:analysis
chemical_list: D001560:Benzidines; D011506:Proteins; C021758:3,3',5,5'-tetramethylbenzidine; D003101:Collodion