Protein Analysis FAQ

• • Why Are the Marker Bands Curved in SDS-PAGE While Protein Bands Appear Normal

  If the marker (also referred to as molecular weight standard or molecular weight ladder) exhibits a curved or hook-like appearance during SDS-PAGE electrophoresis, while the protein bands remain unaffected, several factors may be responsible: 1. Issues with the Electrophoresis Apparatus Poor contact between electrodes or an unstable power supply can result in uneven current distribution, which may interfere with the migration of the marker. In such cases, the electrophoresis system and power source should..

• • What Should Be Done If Sample Leakage Occurs at the Loading Wells During SDS-PAGE Protein Electrophoresis

  If sample leakage occurs from the loading wells during SDS-PAGE, several potential causes should be considered: 1. Gel Preparation Issues Improper gel preparation may lead to enlarged or irregularly shaped wells, increasing the risk of sample leakage during the loading process. 2. Inadequate Loading Technique Lack of proficiency in pipetting techniques may result in accidental leakage while introducing the sample into the wells. 3. Excessive Sample Volume Applying a sample volume that exceeds the well .....

• • What Methods Can Be Used to Measure Intracellular Cystine Levels

  To measure intracellular cystine levels, the following analytical techniques are commonly employed: 1. High-Performance Liquid Chromatography (HPLC) Sample preparation: Cystine is first isolated from cells, typically through cryogenic grinding followed by acidic or alkaline hydrolysis to lyse the cells and release intracellular contents. Derivatization: To enhance detection sensitivity, cystine is often derivatized using reagents such as o-phthaldialdehyde (OPA). Chromatographic analysis: The sample is.....

• • How Can Mass Spectrometry Be Used to Analyze or Predict Protein–Protein Interactions

  Mass spectrometry (MS) has emerged as a powerful tool for analyzing proteins and their interactions. When combined with protein purification techniques—such as co-immunoprecipitation or affinity purification—MS enables the accurate identification of protein complex components. The following outlines the fundamental steps involved in using mass spectrometry to analyze and predict protein–protein interactions: 1. Sample Preparation Proteins are first extracted from cells or tissues. To identify interaction...

• • Why Does a Custom Polyclonal Antibody Yield a Single Band in Western Blot but Fail to Detect the Target Protein

  When a custom polyclonal antibody produces a distinct single band in Western blot (WB) analysis, yet no corresponding target band is observed in the protein sample—accompanied instead by non-specific bands—several potential causes should be considered: 1. Insufficient Antibody Specificity The antibody may exhibit limited specificity, leading to binding with off-target proteins and the appearance of non-specific bands. This could result from cross-reactivity or non-specific interactions. In such cases.......

• • Why Can't Thylakoid Complex Proteins Be Separated by Blue Native PAGE

  Blue Native PAGE (BN-PAGE) is a widely employed technique for resolving protein complexes, particularly membrane proteins and high-molecular-weight assemblies. However, the failure to separate thylakoid supercomplexes by BN-PAGE can arise from several technical and biochemical factors: 1. Suboptimal Sample Preparation The integrity of protein complexes is critically dependent on proper sample preparation. Premature degradation or aggregation of protein assemblies prior to electrophoresis can impede.........

• • What Could Cause Peak Overlapping in Results When Contamination Is Unlikely and Purification Was Carefully Performed

  In scientific analysis, peak overlapping refers to the presence of multiple unresolved peaks in analytical results, often indicating complications in the analytical process. Due to the lack of specific experimental context, the following outlines several general factors that could potentially cause peak overlapping: 1. Sample Preparation Improper sample preparation may result in heterogeneous sample composition, leading to overlapping peaks during analysis. 2. Selection of Analytical Method The chosen......

• • How to Determine the Sites of Protein Post-Translational Modification

  Determining the sites of protein post-translational modifications (PTMs) typically involves an integrated approach that combines experimental techniques with bioinformatics tools. The following methodologies are commonly employed: 1. Mass Spectrometry (MS) Mass spectrometry is the most widely used technique for identifying PTM sites. Proteins are enzymatically digested (e.g., using trypsin) to produce peptides, which are subsequently analyzed by a mass spectrometer. The resulting spectra provide ........

• • Is Cell Lysis the Next Step After Protein Crosslinking? What Is the Best Lysis Buffer to Use

  Protein crosslinking is a commonly employed technique in cell and molecular biology aimed at stabilizing protein–protein interactions within cells. Typically, cell lysis follows crosslinking as the next step. The purpose of lysis is to disrupt the cellular membrane, thereby releasing proteins and other macromolecules. The choice of lysis buffer depends on the cell type and the objectives of subsequent experiments. Below are several commonly used lysis buffers and the scenarios in which they are most .......

• • Q&A of Protein Sequencing

  Q1: Does protein sequencing require the protein to remain active? A1: No. Protein sequencing focuses on determining the amino acid sequence, not the biological activity of the protein. Whether using Edman degradation or mass spectrometry, the functional state of the protein has no impact on sequencing accuracy. In fact, most sequencing workflows—including reduction, alkylation, digestion, and ionization—disrupt the protein’s native structure and eliminate biological activity. What matters