Protein Analysis FAQ

• • How Is Two-Dimensional Electrophoresis Applied in Proteomics?

  Two-dimensional electrophoresis (2-DE) is an essential technique in proteomics, enabling high-resolution separation and analysis of complex protein mixtures. Its applications span multiple areas of protein research, including the following:   Protein Separation and Identification Two-dimensional electrophoresis allows for protein separation based on isoelectric point (pI) in the first dimension and molecular weight in the second. This technique can resolve thousands of proteins simultaneously, facilit......

• • How to Obtain Peptides from Alkaline Protease Hydrolysis of Tartary Buckwheat Protein?

  How to Obtain Peptides from Alkaline Protease Hydrolysis of Tartary Buckwheat Protein? Which Is More Effective: pH Adjustment, TCA Precipitation, or Centrifugation?   Once protein is extracted from tartary buckwheat, enzymatic hydrolysis using alkaline protease is commonly employed to obtain peptides. Following hydrolysis, peptide recovery and purification require further processing. Commonly used methods include adjusting the pH to the isoelectric point, trichloroacetic acid (TCA) precipitation, and ......

• • What Are the Differences Between Triple Quadrupole and Time-of-Flight Mass Spectrometers?

  Principle Differences The triple quadrupole mass spectrometer (TQMS) is a type of tandem mass spectrometer comprising three quadrupole mass filters (Q1, Q2, and Q3). Q1 and Q3 function as mass selectors, while Q2 serves as a collision cell. The sample first passes through Q1, which selects specific precursor ions. These ions then undergo collision-induced dissociation (CID) in Q2, generating fragment ions, which are subsequently filtered by Q3 before reaching the detector. This technique offers high s......

• • Why Is the Target Protein Signal Lower Than Expected in Western Blot Experiments?

  Several factors can lead to a lower-than-expected target protein signal in Western Blot experiments:   Protein Extraction Issues 1. Incomplete Cell Lysis Inefficient cell lysis may result in incomplete protein extraction. Consider optimizing the lysis method by using more effective lysis reagents or employing ultrasonic disruption.   2. Protein Degradation Proteins may degrade during extraction due to enzymatic activity. To prevent this, add protease inhibitors during extraction and minimize handling ......

• • What Are the Methods for Quantifying Protein Expression in a Single Cell?

  Quantifying the expression of a specific protein in a single cell is essential for understanding cellular heterogeneity and its functional implications. Several commonly used methods for single-cell protein quantification include:   Single-Cell Immunofluorescence This fluorescence-based method uses labeled antibodies to detect and quantify specific proteins in individual cells. Target proteins are visualized under a fluorescence microscope, and image analysis software is used to quantify fluorescence ......

• • What Are the Main Methods for Protein Separation and Purification and Their Underlying Principles?

  Below are some commonly employed methods for protein separation and purification, along with their fundamental principles:   Salting Out This method involves gradually increasing the salt concentration (e.g., ammonium chloride) to induce protein precipitation. Since the solubility of proteins varies depending on salt concentration, selective precipitation enables partial purification.   Electrophoresis Proteins are separated based on their mobility in an electric field. Common electrophoretic techniqu......

• • What Software Can Be Used to Calculate the Proportions of Different Secondary Structures from Circular Dichroism Data?

  Circular dichroism (CD) is a powerful technique for studying protein secondary structures, and several software tools are available to calculate the proportions of different secondary structures based on CD data:   DICHROWEB DICHROWEB is an online tool used to analyze circular dichroism data and predict the secondary structure of proteins. It relies on multiple established secondary structure databases and algorithms to provide predictions based on the input data.   K2D3 K2D3 is another widely used on......

• • How Are Primers Designed for Site-Directed Mutagenesis in Protein Interaction Studies?

  Site-directed mutagenesis is a widely used approach for introducing specific point mutations at amino acid sites in protein interaction studies. This process requires the design of mutagenic primers to introduce targeted changes into the DNA sequence using polymerase chain reaction (PCR)-based methods.   The following steps are essential for designing primers for site-directed mutagenesis:   1. Selection of the Mutation Site Identify the specific mutation (substitution, insertion, or deletion) to be i......

• • What Are the Common Methods for Protein Purification?

  Protein purification is a fundamental technique used to isolate and purify a protein of interest, aiming to obtain samples with both high quantity and purity.   Common methods of protein purification include:   Precipitation This technique relies on the differences in protein solubility under various conditions, such as changes in pH, electrolyte concentration, temperature, and the presence of organic solvents. Precipitants like ammonium sulfate, acetic acid, and PEG are commonly used to achieve prote......

• • How to Identify Key Targets in Protein-Protein Interaction Networks?

  In protein-protein interaction (PPI) networks, identifying key targets is essential for elucidating biological processes, understanding disease mechanisms, and developing potential therapeutic strategies. Below is a systematic approach to identifying key targets within PPI networks:   Construction of the PPI Network Retrieve protein-protein interaction data from databases (e.g., STRING, BioGRID) and bolster its reliability through experimental validations (e.g., yeast two-hybrid, mass spectrometry ana......