title: A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB.
abstract: Western blotting is a widely used analytical technique for detection of specific protein(s) in a given sample of tissue/cell homogenate or extract. Both chemiluminescence (CL) and colorimetric detections can be used for imaging Western blots. Colorimetric substrates offer background free, sensitive, and clean imaging results directly on the blotted membrane and provides more accurate profile with respect to prestained marker. However, blots stained with colorimetric substrates cannot be reused since no stripping protocols have been reported for such blots, thus limiting their reuse for detection of another protein. In the present study, for the first time, we report a novel method of stripping Western blots developed with the colorimetric substrate TMB for detection of a low-abundant protein and reprobing of these blots after stripping for detection of a more abundant protein through CL procedure. The stripping procedure utilizes a stripping buffer consisting of β-mercaptoethanol, SDS, and Tris-HCl and a washing buffer consisting of PBS added with 0.1% Tween-20 involves a series of steps and facilitates accurate detection of the second protein (i.e., more abundant protein) in the stripped blot through CL. The protocol is reproducible and facilitates saving of precious clinical samples, in addition to saving cost and time as compared to the existing procedures.
authors.affiliation: Central Drug Research Institute, Lucknow, 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
authors.initials: M
authors.lastname: Lal Bhatt
authors.forename: Monika
authors.initials: M
authors.lastname: Sachdev
mesh_terms: D001560:Benzidines / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D003124:Colorimetry / Q000379:methods*; D017743:Equipment Reuse; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008623:Mercaptoethanol; D008956:Models, Chemical; D011506:Proteins / Q000032:analysis; D012967:Sodium Dodecyl Sulfate
chemical_list: D001560:Benzidines; D014408:Biomarkers, Tumor; D002021:Buffers; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; C021758:3,3',5,5'-tetramethylbenzidine; D008623:Mercaptoethanol