Protein Analysis FAQ

• • Why Is MRM the Preferred Method for Accurate Quantification in Mass Spectrometry?

  To accurately understand why MRM is the most effective method for quantification, it is essential to define the relationship among SIM (Selected Ion Monitoring), SRM (Selected Reaction Monitoring), and MRM (Multiple Reaction Monitoring). SIM and SRM can be regarded as specific applications within the broader MRM framework. SIM is primarily employed in single quadrupole mass spectrometers, whereas SRM and MRM are mainly utilized in triple quadrupole mass spectrometers. In many contexts, SRM and MRM are......

• • Why Do Western Blot Bands Appear Inconsistent for Proteins Extracted from the Same Batch?

  When conducting a Western blot experiment, inconsistent band patterns from the same batch of extracted proteins may arise due to the following factors:   Variations in Electrophoresis Conditions Fluctuations in voltage or current can impact protein migration and separation.   Gel-Related Issues Defects in the SDS-PAGE gel, such as uneven polymerization or prolonged storage, may affect protein resolution.   Transfer Process Irregularities Non-uniform current distribution or improper transfer time and v......

• • What Are the Effects of Polarity Reversal During Protein Electrophoresis?

  In protein electrophoresis, reversing the polarity of the power supply leads to the reverse migration of proteins. Under normal conditions, proteins migrate from the cathode (negative electrode) to the anode (positive electrode) due to the applied electric field. However, if the polarity is reversed, the direction of the electric field is also inverted, causing proteins to migrate in the opposite direction. As a result, proteins may move out of the gel and diffuse into the buffer, preventing proper se......

• • 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...