Smeared Bands

Particulate in the sample: Standard loading buffers are often unable to completely dissolve complex samples-  fragments of bacterial cell walls, for example, can be carried over into the well.  Particulate material in the well blocks proteins from entering the gel and can bind proteins, allowing them to “bleed” into the lane over time, causing characteristic…

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Blotches on Gel

EC895

This gel can be recovered in most cases by fully destaining in methanol:water: acetic acid, followed by a fresh round of staining.  In severe cases, the deposits may be removed by gently spraying the gel surface with alcohol.  Blotches can be caused by overhandling of the gel, or by handling the gel without gloves.  This…

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Uneven Staining

EC895

Uneven staining is almost always the result of insufficient agitation during staining.  The gel is initially more dense than the staining solution and will tend to sink to the bottom of the dish.  Some portions of the gel will have stains under them, others will not, and the gel will show darker and lighter areas…

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Faint Bands, High Background

EC887

A high background that obscures the bands, or in combination with fainter than usual bands, indicates dye binding to the gel matrix, or contamination of the matrix with a dye-binding material (most often a protein). Coomassie Blue R-250: Destaining time too short:  It takes hours for the dye to completely diffuse out of the gel…

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Faint bands, low background

EC890

SDS in the staining solution:  Most users save and re-use their staining solutions.  This is fine for a couple of cycles, but over time enough SDS elutes from the gels to accumulate to significant levels in the staining reagent.  SDS interferes with the binding of the dye to the proteins, resulting in bands that are…

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Coomassie Blue Stain- Troubleshooting

EC895

If your gel doesn’t look like this one, click on the problem below to find the solution: Faint bands on a low background Learn More Faint bands on a high background Learn More Uneven Staining Learn More Dark Blotches on Gel Learn More Smeared or blurred bands Learn More The ProtoGel Sample Prep Kit gives…

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Mechanism of Immunostaining

Basic method of immunostaining

The highly specific binding interaction between antibodies and their unique antigens has been exploited to create sensitive and specific detection systems for proteins. An antibody can be raised and/or purified “against” (i.e. binding to) a single “epitope” (area bound by the antibody) on a larger “antigen” (a molecule containing many epitopes). When antibody and antigen…

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Method for Western Blotting

ELECTROPHORESIS Prepare and run an SDS PAGE gel. Select a gel percent which will give the best resolution for the size of antigen being analyzed (if known). If the size is not known, a 12% gel is a good starting point. Load enough protein to provide 0.1-1ng of antigen per well. It is generally advantageous…

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Overview of Western Blotting

Western blotting apparatus

Proteins can also be detected immunologically following electrophoresis, a technique known as Western blotting. This method relies on the fact that most epitopes (sites recognized by antibodies, generally comprising several amino acids) are still recognizable following denaturing of the protein with SDS and binding to the surface of a membrane. The Western Blotting apparatus. Proteins…

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Enzyme Linked Immunosorbent Assay (ELISA)

Variations of ELISA

One of the most straightforward applications of immunological detection is the ELISA or enzyme-linked immunosorbent assay. In the simplest system, the bound antigen is probed with antibodies that carry covalently attached enzyme molecules. Antibody binding immobilizes the enzyme in the vicinity of the bound antigen, allowing detection of the antigen. Variations include a competition ELISA…

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