Protein Analysis FAQ
-
• How to Detect Protein Phosphorylation Without Site Information or Available Antibodies
Detecting protein phosphorylation without prior knowledge of the modification sites or access to site-specific antibodies can be challenging. However, several alternative strategies are available: Mass Spectrometry (MS) 1. Protein Digestion Initially, proteins are enzymatically cleaved into peptides using proteases such as trypsin. 2. Enrichment of Phosphorylated Peptides Phosphorylated peptides are selectively enriched using techniques like Immobilized Metal Affinity Chromatography (IMAC) or titanium......
-
• Why Blue Dyes in White Balance Sometimes Appear as Thin Bands and Sometimes as Diffuse Broad Bands
This issue pertains to the control of electrophoretic conditions and staining performance in Western blot experiments. If you observe that the blue dye occasionally forms sharp, narrow bands and at other times appears as diffuse, broad bands, several factors may be responsible. The following are potential causes and their corresponding solutions: 1. Improper Sample Preparation Inadequate staining or uneven visualization of protein samples can result in dye diffusion. Moreover, using samples with ...........
-
• What Is the Role of Co-Immunoprecipitation
Co-immunoprecipitation (Co-IP) is a widely used experimental technique for detecting and isolating specific proteins along with their interacting partners. It plays a critical role in biopharmaceutical development and biotechnology, as it provides insight into the mechanisms of protein-protein interactions and thereby facilitates understanding of disease initiation and progression. The fundamental principle of Co-IP involves leveraging the high specificity of antibodies to bind target proteins, allowing....
-
To assess whether a protein's activation state is modulated through phosphorylation or dephosphorylation, the following approaches can be employed: 1. Bioinformatics-based Prediction Bioinformatics tools—such as GPS 2.0, NetPhos, among others—can be used to predict potential phosphorylation sites within the protein sequence. When these predicted sites are located within functional domains, particularly within evolutionarily conserved regions in diploid yeast, it is highly suggestive that phosphorylation....
-
• What Are the Specific Experimental Methods for Cross-Linking Protein Interaction Analysis
Cross-linking is an experimental strategy widely employed to investigate protein-protein interactions. By applying cross-linking reagents—typically chemical agents—to covalently link two or more interacting protein molecules, these interactions are stabilized, facilitating subsequent detection and characterization. The following outlines several commonly used experimental approaches for cross-linking-based protein-protein interaction analysis: Chemical Cross-Linking 1. Sample Preparation Target cells or....
-
• What Software Is Available for De Novo Sequencing in Mass Spectrometry-Based Sequencing
Mass spectrometry-based sequencing is a method for identifying protein sequences. De Novo Sequencing refers to the reconstruction of amino acid sequences from mass spectrometry data without reference to known protein sequence databases. Within this context, De Novo Sequencing plays a critical role in identifying unknown proteins and post-translational modification sites. The following are commonly used software tools for performing De Novo Sequencing on mass spectrometry data: 1. PEAKS PEAKS is a widely ...
-
• How to Handle Samples After Adding Crosslinkers in Protein Crosslinking
In experiments analyzing protein–protein interactions via crosslinking methods, cells are commonly treated with chemical crosslinkers such as formaldehyde. Following crosslinker treatment, it is essential to quench the crosslinking reaction. This can typically be achieved by adding Tris buffer, glycine, or by rapidly cooling the cells to 4°C. After crosslinking, the cells may be either flash-frozen for storage or processed immediately for lysis. For example, when glycine is used to terminate the ........
-
• Why Does Protein Electrophoresis Move Toward the Negative Electrode
In protein electrophoresis, whether a protein migrates toward the negative or positive electrode depends on its charge in the electrophoresis buffer. A protein’s charge is determined by the charges of its amino acid residues, which vary with pH. The charge of a protein is influenced by the solution's pH and its isoelectric point (pI). The isoelectric point (pI) is the pH at which a protein has a net charge of zero. Below this pH, the protein carries a positive charge; above this pH, it carries a negative...
-
• Why Is It Necessary to Detect Phosphorylated Proteins in WB When Detecting Proteins
Western Blot (WB) is a key technique in biological research used to detect specific proteins in a sample. A common question is why researchers need to specifically detect phosphorylated proteins. The answer lies in the fact that protein phosphorylation is a crucial biological event that significantly alters protein function and activity. Here’s a detailed explanation: What is Western Blot (WB)? Western Blot is a widely used method for identifying and quantifying specific proteins in cells or tissues. The...
-
• When to Use Western Blot vs ELISA for Protein Detection
Western Blot and ELISA are two common methods for protein detection, each with its own advantages and use cases. Western Blot 1. Use Case Typically used to detect the presence, relative expression levels, and specific modifications of proteins, such as phosphorylation or methylation. Provides information on molecular weight and immunoreactivity. 2. Procedure Involves protein extraction, separation (usually via SDS-PAGE), transfer to a membrane, antibody binding, visualization, and analysis.
How to order?
