Protein Analysis FAQ

• • What Are the Possible Causes of Protein Band Degradation in Electrophoresis?

  If protein bands appear degraded during electrophoresis, several factors may contribute to this issue:   Improper Protein Sample Handling Proteins may undergo degradation due to protease activity during extraction, storage, or handling. To prevent this, protease inhibitors should be added during extraction, and all procedures should be conducted at low temperatures to minimize proteolysis.   Suboptimal Electrophoresis Conditions The stability of proteins can be affected by electrophoresis parameters s......

• • Is It Appropriate to Use Monoclonal and Polyclonal Antibodies for Different Proteins in Immunofluorescence and Western Blot?

  The selection between monoclonal and polyclonal antibodies is not a matter of standardization but rather an essential aspect of experimental design. Choosing different types of antibodies depending on the experimental objectives and conditions is scientifically valid. The critical factor is to ensure that the selected antibodies have been properly validated and can specifically detect the target protein under the applied experimental conditions.   Considerations for Selecting Monoclonal or Polyclonal ......

• • How to Effectively Detect HIF-1α Protein in Western Blot Analysis?

  Detecting the target protein (e.g., HIF-1α) in a Western blot experiment requires the following steps:   Cell Culture and Treatment 1. Cell Culturing Select an appropriate cell line, such as human cell lines (e.g., HEK293, HeLa) or mouse cell lines (e.g., NIH/3T3), and culture the cells in the appropriate growth medium.   2. Cell Treatment Treat the cells according to the experimental design to induce or inhibit the expression of the target protein. For example, chemical agents (e.g., drugs) or physic......

• • How to Determine the Number of Unique Peptides in MaxQuant Analysis Results?

  MaxQuant is a widely used software for quantitative proteomics that processes mass spectrometry data to identify proteins and peptides.   In MaxQuant’s output files, information on unique peptides can be found in "peptides.txt," which contains detailed data for each identified peptide.   Each row in "peptides.txt" represents an identified peptide. The "Sequence" column lists the peptide’s amino acid sequence, the "Length" column specifies its length, and the "Modifications" column records any post-tra......

• • How to Improve Band Clarity for 220 kDa Proteins in Western Blot?

  Achieving clear bands for large proteins (e.g., 220 kDa) in Western Blot (WB) can be challenging due to migration and transfer limitations. The following strategies can help optimize WB conditions for large proteins:   1. Gel Selection Use a low-percentage polyacrylamide gel (e.g., 6% or 8%) to increase pore size and enhance the resolution of large proteins.   2. Electrophoresis Conditions (1) Run the gel at a low voltage (e.g., 80–100V) to prevent excessive migration speed and minimize gel overheatin......

• • Why Do SDS-PAGE Results Show Only Reduced Bands and No Non-Reduced Bands

  SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a widely employed technique for protein analysis. Running SDS-PAGE under both reducing and non-reducing conditions facilitates the evaluation of protein structure and function. The absence of non-reduced bands despite the presence of reduced bands may be attributed to several factors: 1. Protein Structure Under non-reducing conditions, disulfide bonds remain intact, preserving the native conformation of the protein. If a protein is.....

• • Why Should Samples Not Be Loaded on Both Sides When Determining Protein Molecular Weight by SDS-PAGE

  SDS-PAGE is a widely used technique for protein separation and molecular weight determination. In this method, sodium dodecyl sulfate (SDS) binds to proteins, conferring a uniform negative charge, thereby allowing migration toward the anode under an applied electric field. However, the distribution of the electric field in the electrophoresis buffer can be influenced by sample loading positions. When samples are loaded on both sides of the gel, local ion concentration in the buffer is altered, leading to...

• • How to Identify the Interacting Proteins of a Specific Protein in Detail

  To systematically identify the interacting proteins of a specific protein, various experimental strategies can be utilized. The most commonly employed protein-protein interaction (PPI) analysis techniques include: 1. Co-Immunoprecipitation (Co-IP) Co-IP is a widely used method for studying direct and stable protein-protein interactions. It employs specific antibodies to precipitate a target protein along with its interacting partners, which are subsequently analyzed using mass spectrometry or Western.......

• • How to Easily Measure Total Sugar and Protein Content in Plants

  Determination of Total Sugar Content 1. Anthrone Method This is a widely adopted method for the determination of total sugar content. Plant samples are first subjected to appropriate pretreatment and extraction, followed by reaction with the anthrone reagent. The reagent interacts with sugars to form a blue-green chromophore, whose absorbance is measured to quantify the sugar concentration. 2. Phenol-Sulfuric Acid Method This is another widely used technique for quantifying total sugars. Following .......

• • Do Proteins With Smaller Relative Molecular Mass Migrate Faster in SDS-Polyacrylamide Gel Electrophoresis

  SDS-PAGE employs an electric field to separate proteins based on their migration through a polyacrylamide gel matrix. SDS (sodium dodecyl sulfate) is an anionic surfactant that binds uniformly to proteins, denaturing them and imparting a consistent negative charge. As a result, protein migration becomes independent of their native charge and is primarily determined by their size (molecular mass). In SDS-PAGE, the migration rate of proteins is chiefly governed by their molecular mass. Proteins with smaller