nonspecific Protocols

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature.  Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts.  Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags.  Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. - [Read Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies.  Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins.  It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses FAM-(6-carboxy-fluorescein) or JOE-(6-carboxy-4', 5' -dichloro-2',7' -dimethoxy-fluorescein) labeled LUX (Light Upon eXtension) primers, which can quantify 100 or fewer copies of the target DNA in a background of nonspecific templates, over a broad dynamic range of less than 100-107 copies.  It uses uracil deglycosylase (UDG) to minimize the risk of carryover contamination, and includes a melting curve analysis of the product. - [Read Real-Time PCR Protocol]

Category: Bioinformatics

Searches are not constrained for only tryptic peptides, and indexed databases (databases only containing tryptic peptides) are not used. In cases where there are very complex mixtures, such as cell lysates, nonspecific cleavages can occur. Therefore, nontryptic peptides would be missed in the database search. - [Read The Use of Mass Spectrometry in Proteomics: Database Searching]

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Troubleshooting the immunoprecipitation procedure Roche. Tagged proteins not visible or only weakly visible on Western blot. High background or many nonspecific bands on gel or blot membrane. - [Read Troubleshooting the immunoprecipitation procedure IP and IB - Roche]