Protein Analysis FAQ

• • What Analytical Techniques Are GC, GC/MS, LS, LC/MS, ICP-MS, IR, UV, and NMR? What Are They Primarily Used for?

  This section provides an overview of various analytical techniques used for compound separation, identification, and quantification.   Gas Chromatography (GC) 1. Principle GC is primarily used for the separation and analysis of volatile compounds based on their interaction with a stationary phase under controlled temperature conditions.   2. Applications Used to determine the composition and relative abundance of volatile organic compounds in complex mixtures.   Gas Chromatography–Mass Spectrometry (G......

• • How Can Potential Modifications of a Protein Be Identified, Such as Phosphorylation or Acetylation in Different Conditions?

  Identifying potential protein modifications is typically achieved through a combination of bioinformatics analysis and experimental techniques. The commonly used approaches include:   1. Bioinformatics-Based Prediction Online databases and computational tools such as UniProt, PhosphoSitePlus, and PROSITE provide information on known post-translational modifications (PTMs) and predict potential modification sites.   Comparative sequence analysis can help identify conserved modification sites by analyzi......

• • What Techniques Are Used to Analyze Protein Structure?

  Protein structure can be studied at different hierarchical levels, including primary structure (amino acid sequence), secondary structure (local motifs such as α-helices and β-sheets), tertiary structure (the three-dimensional conformation of a single protein molecule), and quaternary structure (complexes formed by multiple interacting subunits). A variety of techniques are available for structural analysis, each with distinct advantages and limitations. The most commonly used methods include:   1. X-......

• • Why Is the FLAG Tag Not Detectable by Western Blot Despite a Strong Band Observed with the Target Antibody?

  When expressing FLAG-tagged proteins, if a strong band is detected using the target antibody but the FLAG tag is not detected using the tag-specific antibody via Western blot (WB), several factors may contribute to this issue:   1. Insufficient Specificity or Reduced Activity of the FLAG Antibody The FLAG tag antibody may have insufficient specificity or reduced activity, which could result from the antibody itself, expiration, or degradation during storage.   2. Restricted Accessibility of the FLAG T......

• • How Can the Concentration of a Sample Be Determined Using Gas Chromatography Based on Peak Area and Retention Time?

  Gas chromatography (GC) is a widely employed analytical technique that is used to determine the concentration of a sample based on the retention times and peak areas of its components. The procedure is as follows:   Establishing a Standard Curve First, chromatograms for standard solutions of varying concentrations are acquired, with the retention times (tR) and peak areas of each component recorded. A standard curve is then plotted by correlating the concentration of each standard solution with its co......

• • Why Does the Highest Observed Mass-to-Charge Ratio in Mass Spectrometry Correspond to the Relative Molecular Mass

  In mass spectrometry, the highest observed mass-to-charge ratio (m/z) refers to the maximum m/z value detected in a mass spectrum. A key question in molecular mass determination is why this highest observed m/z corresponds to the relative molecular mass of the analyte. The following discussion will clarify this relationship step by step.   1. Fundamental Principles of Mass Spectrometry Mass spectrometry is an analytical technique that identifies ions based on their mass-to-charge ratios by converting ......

• • Why Is the Protonated Molecular Ion Observed as [M+H]⁺ in ESI Mass Spectra?

  Electrospray ionization (ESI) is a widely employed soft ionization technique in mass spectrometry, particularly for the analysis of biomolecules such as proteins, peptides, and nucleic acids. In ESI, the sample solution is dispersed through a charged electrospray needle, generating fine droplets that undergo ionization under a high-voltage electric field.   Why [M+H]⁺ 1. Protonation Mechanism In the electrospray process, analyte molecules in the droplets readily capture protons (H⁺), primarily due to ......

• • What Is the Standard Protocol for Glutaraldehyde Cross-Linking? Is a Detailed Protocol Available?

  Glutaraldehyde cross-linking is a widely used technique for cross-linking, fixing, and stabilizing proteins and other biological macromolecules. The standard protocol consists of the following steps: Sample PreparationWash the sample (e.g., cells or tissues) with PBS (phosphate-buffered saline) or another suitable buffer to remove residual media, debris, or other contaminants. Preparation of Glutaraldehyde SolutionDilute glutaraldehyde to the desired concentration (typically 0.1% to 2.5%) in PBS o......

• • What Is the Significance of Determining a Protein’s Isoelectric Point

  The isoelectric point (pI) of a protein is the pH at which the protein has a net charge of zero under specific conditions. Determining the isoelectric point is critical for understanding various physicochemical properties of proteins and has several important implications: Understanding the Charge State of a ProteinThe charge state of a protein significantly influences its solubility, stability, interactions, and functionality. Variations in pH alter the protein’s net charge, affecting its solubilit......

• • Requesting Assistance From an Expert in MRM Mass Spectrometry

  1. What is MRM? Multiple Reaction Monitoring (MRM) is a quantitative mass spectrometry technique that enables highly sensitive and selective detection of target molecules by monitoring specific precursor-product ion transitions. 2. Principle of MRM MRM operates on a triple quadrupole mass spectrometer. In this mode, the first quadrupole (Q1) selectively filters a precursor ion of interest. The ion then undergoes fragmentation via collision-induced dissociation (CID) in the second quadrupole (q2). Fina......