Protein Analysis FAQ

• • What Are the Possible Reasons for the Molecular Weight of Proteins Detected by SDS-PAGE Being Higher Than Expected

  When analyzing proteins using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), the observed molecular weight may exceed the expected value due to several factors: 1. Protein Aggregation Under certain conditions, proteins can form dimers or larger aggregates. This may result from the intrinsic biochemical properties of the protein or from specific conditions during sample preparation, such as variations in pH, temperature, and ionic strength. If aggregation occurs, the migration rate....

• • How to Analyze the Glycosidic Bond Structure Using GC-MS Detection

  Mass spectral analysis is the primary approach for interpreting glycosidic bond structures when using gas chromatography-mass spectrometry (GC-MS). The following key steps should be considered for result interpretation: 1. Analysis of GC Chromatogram Peaks The peaks in the GC chromatogram represent different compounds and must be identified. Each peak corresponds to a mass spectrum, which provides information on the mass distribution of the detected compound. 2. Detailed Mass Spectral Analysis Molecular....

• • Why Are the Protein Bands Unclear in SDS-PAGE Analysis of Plant Proteins

  SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a widely used technique for protein analysis. However, when applied to plant proteins, unclear or undetectable bands may arise due to the following factors: 1. Interference from Impurities in Protein Extraction Plant cells contain various secondary metabolites and contaminants, such as polysaccharides and phenolic compounds, which may interfere with protein migration and resolution in SDS-PAGE. 2. Protein Degradation The absence of.....

• • SDS-PAGE Measures Protein Molecular Weight: How Is Migration Distance Measured

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for determining protein molecular weight. Following SDS-PAGE, the molecular weight of proteins can be estimated by measuring their migration distance within the gel. The migration distance of proteins can be determined using image analysis software (e.g., ImageJ) or a ruler by measuring the distance from the sample well to the protein band. Estimating Protein Molecular Weight Using Migration Distance: .......

• • How to Remove the SUMO Tag After Purification

  The Small Ubiquitin-like Modifier (SUMO) tag is a widely utilized fusion tag that facilitates protein solubility and stability. It can be specifically cleaved by SUMO-specific proteases, such as SENP1 or SENP2, to yield the target protein in its untagged form. The standard protocol for SUMO tag removal consists of the following steps Expression and Purification of SUMO-Tagged Protein The target protein is expressed using a SUMO-fusion expression system, commonly in Escherichia coli, and subsequently ......

• • What Is the Principle of Separating Proteins by Gel Chromatography

  Gel chromatography (also referred to as gel permeation chromatography, gel filtration chromatography, or molecular sieve chromatography) is a widely used technique for protein separation and purification. Its principle is based on differences in the molecular size and shape of proteins, which govern their interaction with the gel medium. The gel medium utilized in gel chromatography is typically composed of materials such as polyacrylamide or agarose, which form a three-dimensional network structure........

• • What Is the Essential Difference Between the Top-Down and Bottom-Up Approaches in Proteomics Analysis

  Mass spectrometry is a fundamental technology in proteomics research. In the field of protein mass spectrometry, two primary analytical approaches are employed: the Top-down and Bottom-up strategies. These approaches fundamentally differ in how they process and analyze protein samples. Top-Down Approach In the Top-down approach, intact proteins are directly introduced into the mass spectrometer and subsequently fragmented within the instrument. This means that the analysis begins with the entire protein....

• • What Is Protein Characterization

  Protein characterization encompasses the systematic analysis of the molecular weight, sequence, structure, function, interactions, and other relevant properties of protein molecules. The primary objective is to achieve a comprehensive understanding of protein properties and functions, thereby elucidating their roles in biological processes, including metabolic pathways and signal transduction. This process typically integrates a variety of experimental and computational methods to achieve a thorough .......

• • Why Does the Coloration of Thin-Layer Chromatography Appear Blurry

  Thin-layer chromatography (TLC) is a widely used technique for the separation and identification of compounds in laboratories. However, in certain cases, the visualization of spots may appear unclear. This issue can arise due to various factors, some of which are outlined below: 1. Improper Sample Application Unsteady hands or incorrect handling during sample application can result in poorly defined spots on the TLC plate. It is recommended to apply samples with precision using appropriate tools, such as...

• • Criteria for Identifying the Molecular Ion Peak in Mass Spectra

  The identification of the molecular ion peak (M⁺ peak) in mass spectra is primarily based on the following criteria: 1. Mass-to-Charge Ratio Considerations The molecular ion peak generally corresponds to the highest mass-to-charge ratio (m/z) in the spectrum, as it represents the intact molecule prior to fragmentation. 2. Nitrogen Rule According to the nitrogen rule, compounds containing an even number of nitrogen atoms tend to exhibit a molecular ion peak with an even m/z value, whereas those with an odd..