Protein Analysis FAQ

• • 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...

• • Why Do Large Molecular Weight Protein Bands Appear Wavy in Western Blot

  In proteomics research, Western Blot is a common method for measuring protein expression levels. A common issue observed in Western Blot experiments is the wavy appearance of large molecular weight protein bands (also known as the "smile effect"). This can be caused by several factors: 1. Electrophoresis Conditions (1) Voltage Settings: If the electrophoresis voltage is too high, it may cause proteins to migrate too quickly, leading to wavy bands. (2) Electrophoresis Time: Both overly long and short........

• • 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 Detect Protein Expression in Cells During Experiments

  To detect a protein in cell experiments and determine whether it is expressed, various techniques can be used: 1. Western Blot A common method to detect proteins. First, separate cell proteins by gel electrophoresis, then transfer them to a membrane. Use a specific antibody to detect the target protein. If the target protein is present, the antibody will bind to the corresponding spot on the membrane, forming a band. The presence of this band confirms protein expression. 2. Immunofluorescence Using.........

• • Why Are Genes Not Displayed on the Network Graph When Analyzed with STRING

  STRING is a commonly used protein-protein interaction (PPI) database that allows users to generate PPI networks by inputting a set of protein or gene names. If the genes you entered do not appear on the network graph, there could be several reasons: 1. Gene Name Not Recognized or Mismatched The gene names you entered may be misspelled or use incorrect naming conventions, preventing STRING from recognizing them. 2. Missing PPI Data for Genes While STRING contains a vast amount of PPI data, not all genes.....

• • Why Does High-Performance Liquid Chromatography Data Show Extraneous Peaks

  HPLC data showing extra peaks may stem from several causes. Here are common possibilities and their solutions: 1. Impurities in the Sample Impurities or residual solvents in the sample can lead to contaminant peaks. Purify and treat the sample before injection to minimize impurities. 2. Residual Reagents and Solvents Reagents and solvents left in parts like the column, syringe, or tubing from previous analyses may cause extra peaks. Thoroughly clean the chromatography system before each run.

• • How to Calculate Protein Expression Levels in Proteomics

  In proteomics research, common methods to measure protein expression levels are based on relative or absolute quantification using mass spectrometry data. Common quantification methods include labeling and label-free approaches: 1. Labeling Quantification Proteins or peptides are labeled using isotope tagging techniques, such as SILAC, ICAT, TMT, and iTRAQ. The relative change in protein expression is calculated by comparing the signal intensity of isotope labels in different samples. Log2-fold change......

• • How to Manually Integrate Peak Area on HPLC

  High-performance liquid chromatography (HPLC) is usually equipped with data processing software for real-time or offline chromatographic data analysis. In the data processing software, peak area can be integrated automatically or manually. Below are the general steps for manually integrating peak area in HPLC data processing software: 1. Open Chromatographic Data Open the chromatographic data file that needs manual integration in the data processing software. 2. Select Manual Integration Mode In the........