proteins Protocols

Category: Antibody Protocols: Antibody Production Protocols

Antibodies directed against bacterial and phage-encoded proteins can be removed from sera by incubation with nitrocellulose filters impregnated with lysates of uninfected or phage-infected E. coli. - [Read Removal of Cross-reactive Antibodies from Antiserum: Pseudoscreening Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Li]

Category: Immunology: Immunoprecipitation Protocols

Protocol for RNA co-immunopurification with specific RNA-binding proteins. Includes: Binding to IgG beads; Washing the beads; Elution with TEV-protease; RNA isolation. - [Read RNA co-immunopurification with specific RNA-binding proteins]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: Protein Protocols: Protein Electrophoresis: SDS-PAGE Electrophoresis Protocols

SDS-PAGE: gel electrophoresis of proteins. Very simple protocol for SDS-PAGE. Koshland Lab, Carnegie Institution of Washington, Howard Hughes Medical Institute. - [Read SDS-PAGE]

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for separating proteins using ion-exchange chromatography. Protocol details the practical considerations of an ion-exchange chromatography (IEC) experiment.  The choice of what type of ion exchanger to use, as well as the composition of the buffers used in this experiment, should be determined prior to beginning this protocol. - [Read Separating Proteins Using Ion-Exchange Chromatography Protocol]

Category: Protein Protocols: Protein Precipitation Procols

Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant. Randolph B. Caldwell et al., AEM ASM. - [Read Simple and Reliable Method To Precipitate Proteins from Bacterial Culture Supernatant PDF]

Category: DNA Protein Interactions Protocols

The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment.  The complex is then UV-irradiated, initiating covalent cross-linking with proteins in direct physical proximity to the cross-linking agent.  Extensive nuclease digestion is performed to eliminate uncross-linked DNA and convert cross-linked DNA to a cross-linked, radiolabeled nucleotide "tag." - [Read Site-Specific Protein-DNA Photo-Cross-Linking: Analysis of Structural Organization of Protein-DNA]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the split protein strategy, a single reporter protein/enzyme (firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X & Y. Physical interactions between the two proteins reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assay - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Cells]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), or with tumor models grown from tumor cells stably expressing the complete reporter system. - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice]

Category: Western Blot Protocols: Immunoblot Stains for Total Protein Protocols

Protocol used to determine the position of molecular-weight markers and to ensure that efficient transfer of proteins to the blot has occurred, the total composition of the proteins can be determined by staining the membrane with Ponceau S (Staining Immunoblots for Total Protein Using Ponceau S) or India ink. - [Read Staining Immunoblots for Total Protein Using India Ink Protocol]

Category: Western Blot Protocols: Immunoblot Stains for Total Protein Protocols

Protocol used to determine the position of molecular-weight markers and to ensure that efficient transfer of proteins to the blot has occurred, the total composition of the proteins can be determined by staining the membrane with Ponceau S . - [Read Staining Immunoblots for Total Protein Using Ponceau S Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes how to perform a test separation using a set of protein standards that have been designed to evaluate column performance. - [Read Standard Chromatographic Conditions for RP-HPLC of Proteins Protocol]

Category: Western Blot Protocols: Immunoblot Stripping Protocols

Protocol describes the practice of stripping immunoblots and reprobing to detect multiple proteins on the same blot is a good method to examine two antigens under identical conditions. - [Read Stripping Immunoblots for Reprobing or Storage Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

In the routine method described in this protocol, chylomicron-free plasma is adjusted to 12% (w/v) iodixanol and the sample, essentially fills an approx 3 ml tube for a near-vertical rotor. During the centrifugation VLDL, LDL and HDL particles and also plasma proteins migrate from all parts of the sample to their final buoyant density banding position in the self-forming density gradient. - [Read Subfractionation of Low-Density Lipoprotein (LDL)]

Category: Protein Protocols: Protein Extraction From Tissue

Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs / tissue - [Read Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomic]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection into 293T cells is a convenient way to overexpress and obtain both cellular and extracellular (secreted or membrane) proteins. 293 is a human renal epithelial cell line which is transformed by adenovirus E1A gene product. 293T is a derivative which also express SV40 large T antigen, allowing episomal replication of plasmids containing the SV40 origin and early promoter region. They (both) have the unusual property of being highly transfectable. - [Read Transient Transfection Into 293T Cells Protocol]

Category: Protein Protocols: Protein Electrophoresis

Tricine–SDS-PAGE Protocol and background. Nature. PDF file. Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. –SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification. - [Read Tricine–SDS-PAGE Protocol PDF]

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Troubleshoot Immunoprecipitation. Chemicon. The most common challenge with immunoprecipitation is trying to lower the number and type of background proteins that contaminate the washed immune complexes. Suggestions for decreasing background in IP. - [Read Troubleshoot Immunoprecipitation]

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]

Category: Protein Protocols: Protein Ubiquitination Protocols

Ultimate His-UB Assay for Mammalian Cells. Tansey Lab Protocols. William P. Tansey. PREPARATION OF NI-NTA-AGAROSE, HARVESTING THE TRANSFECTION., ELUTION OF HIS-TAGGED PROTEINS. - [Read Ultimate His-UB Assay for Mammalian Cells]

Category: Protein Protocols: Protein Trafficking

Background and methods to study plant transport. Includes new methods to study protein trafficking in plant cells, includes: Identification of protein sorting pathways in non purified samples; Localization of organelle proteins by isotype tagging/isotype-coded affinity tag; Coupling of chemical genomics and proteomics; Top down mass spectrometry; Compartment-specific markers to aid in the purification of organelles. - [Read Understanding Protein Trafficking in Plant Cells Through Proteomics]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes the use of FLAG-epitope-tagged proteins for both small-scale analysis and large-scale coimmunoprecipitation of interacting proteins. When examining protein interactions, it is sometimes possible to immunoprecipitate an endogenous protein X directly, without using an epitope tag, if antibodies are available. The advantage of examining interactions of endogenously expressed proteins is that these are likely to be physiological and less likely to be an artifact of overexpression. - [Read Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins Protocol]