Ethidium Bromide Staining
Bands in gels stained with ethidium bromide fluoresce under ultraviolet light. The most commonly used stain for detecting DNA/RNA is ethidium bromide. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm. Additionally, it can…
Read MorePost-Electrophoretic Visualization with Nuclistain
National Diagnostics’ Nuclistain offers a significant increase in sensitivity over UV shadowing, along with the convenience of visual staining. With Nuclistain there is no need for UV light. Nuclistain is a blue dye that binds to DNA, revealing blue bands after destaining with water. Nuclistain detects DNA down to levels of 50ng. Note: Nuclistain does not…
Read MoreUV Shadowing
When ultraviolet light strikes a DNA molecule, it may be absorbed, transmitted, or, if a fluorescent dye is present, re-emitted as visible light. Detection of DNA/RNA in solution is generally done by measuring its UV absorbance at 260 nm. This absorbance is due to the ring systems in the nitrogen bases, and can also be…
Read MoreIsoelectric Focusing
Conventional electrophoresis techniques can separate up to 100 different proteins on one run. Typically, cell or tissue extracts contain thousands of proteins, most of which will not be resolved into single bands using a separation based on any one parameter, such as size or net charge. For any one size range, there is a high…
Read MoreImmuno-Electrophoresis / Immuno-Diffusion
When present at approximately equal concentration, antigen and antibody will precipitate as an aggregate. The figure above illustrates a principle underlying the usefulness of immunodiffusion, that with shifts in antibody concentration, a corresponding shift in the region of precipitation will occur. Antibodies are produced by the immune system in response to foreign macromolecules. Each antibody…
Read MoreActivity Stains
Samples to be run on native gels should be prepared in a way that minimizes the denaturation of the proteins. Avoid heat, strong detergents, foaming, and over-dilution. In addition, the activity of endogenous proteases must be kept to a minimum. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A…
Read MoreGel Preparation for Native Protein Electrophoresis
The basic protocols for preparing Native PAGE gels are the same as for discontinuous SDS PAGE gels, substituting non-SDS buffers for those containing SDS, as follows: Casting Native Protein Gels Prepare resolving gel and stacking gel casting solutions The table below gives the formulations for native resolving gels from 6 – 12% as well as…
Read MoreSample Preparation for Native Protein Electrophoresis
Samples to be run on native gels should be prepared in a way which minimizes denaturation of the proteins. Avoid heat, strong detergents, foaming and over-dilution. In addition, the activity of endogenous proteases must be kept to a minimum. Keeping the sample cold and including protease inhibitors will be helpful in this regard. A formulation…
Read MoreNative Protein Electrophoresis
Proteins run on PAGE in the absence of SDS will separate on the basis of their charge to mass ratio. While native (nondenaturing) PAGE does not provide direct measurement of molecular weight, the technique can provide useful information such as protein charge or subunit composition. Native PAGE also has the potential for separating proteins of…
Read MorePeptide Mapping
Peptide mapping involves controlled cleavage of a pure protein with small amounts of a pure protease to generate peptides of characteristic, reproducible sizes. These peptides can be separated on PAGE to produce a “fingerprint” characteristic of the protein. Peptide mapping can map cleavage sites in an unknown protein, or it can identify an unknown protein…
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