Utilizing the same chemistry as black and white photography, silver staining is another highly sensitive method for the visualization of protein bands on electrophoresis gels. Silver ions are reduced to insoluble silver metal granules in the vicinity of the protein molecules. Sufficient silver deposition is visible as a dark brown or black band on the…

The Coomassie dyes (R-250 and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. Coomassie R-250, the more commonly used of the two, can detect as little as 0.1 ug of protein. Though less sensitive, Coomassie G-250 can be…

Fixing both native (left) and SDS denatured (right) proteins with acetic acid and alcohol results in an uncoiling of the peptide chains to produce insoluble complexes and monomers. Fixing (or fixation) is the process whereby proteins are denatured and precipitated in large insoluble aggregates within the gel matrix. Fixation accomplishes several goals. Primarily, fixation prevents…

Autoradiographic Enhancement with Autofluor: The Procedure National Diagnostics’ Autofluor is an extremely sensitive, water based fluorographic enhancer for autoradiography on gels, TLC plates or paper chromatograms. GELS After staining, fix the gel with 5% glacial acetic acid, 5% isopropyl alcohol, and 90% water. Fix for 15 to 20 minutes. Pour off fixing solution and discard…

Autoradiography is the use of X-ray (or occasionally photographic) film to detect radioactive materials. It produces a permanent record of the positions and relative intensities of radiolabeled bands in a gel or blot. Typically, biomolecules are labeled with 32P or 35S, and detected by overnight film exposure. The table below gives the amounts of commonly…

One example of one might attempt alkaline blotting is listed here. Alkaline Blotting: The Procedure Positively charged nylon membranes allow the use of alkaline transfer buffers, which link the nucleic acids to the membrane without UV crosslinking. In some cases, alkaline blotting gives a higher background. Increasing the concentration of blocking reagent will often eliminate…

One possible procedure for Southern blotting can be found here. The Procedure The protocols given are for Genomic Southern Blotting, one of the most sensitive Southern techniques. This set of protocols will give superior results in any standard Southern application. SAMPLE PREPARATION Genomic DNA samples must be extremely pure,with the A260/A280 ratio being equal to…

The Procedure ELECTROPHORETIC SEPARATION RNA samples should be free of contaminating particles and DNA, with an A260/A280 ratio of 1.9-2.0. For highly expressed genes, load 10µg total of RNA. For trace expression detection, load 10µg of poly A+ RNA. Ethidium may be added to the gel (4 µl of 10 mg/ml per 100ml gel =…

A DNA or RNA probe will selectively hybridize with nucleic acid molecules of complementary sequence in a sample. A labeled nucleic acid molecule of known sequence can facilitate detection of any complementary molecules in an unknown sample. This is the basis of the RNase protection assay, and PCR amplification, among other techniques. In theory, labeled…

Silver staining is more sensitive than ethidium bromide for double stranded DNA, and detects single stranded DNA or RNA with no loss in sensitivity. Silver staining relies on the reduction of silver cations to insoluble silver metal by nucleic acids. This chemical reaction is insensitive to the macrostructure of the DNA molecule. Reduced silver grains…