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Updated: Jun 7, 2026
22:45
Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels
Published on: August 22, 2007
Detection and quantitation of radiolabeled proteins in gels and blots
Current Protocols in Toxicology
|October 26, 2010
Abstract:
This unit provides procedures for detecting an quantifying radiolabeled proteins in SDS-polyacrylamide gels or blots. Autoradiography requires fixed, dried gels before exposure to X-ray film. The results may be enhanced by using intensifying screens and/or preflashing the film. Alternatively fluorography with organic scintillant-impregnated gels may be used to enhance signal. The autoradiogram may be quantified by densitometry. Phosphorimaging is a useful alternative to film exposure.
Related Concept Videos
01:15Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
01:22Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as cells...
02:57Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
01:27SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...