Protein Analysis FAQ

• • What is the SEC-HPLC Method for Determining Protein Purity?

  When determining the purity of a protein sample, a commonly employed method involves combining Size Exclusion Chromatography (SEC) with High-Performance Liquid Chromatography (HPLC). The following provides a detailed explanation of the procedure for SEC-HPLC protein purity assessment:   SEC-HPLC is a separation technique based on protein molecular size. It uses a porous gel column where larger protein molecules, unable to enter the gel, flow through the column more quickly, while smaller impurity mole......

• • Why Does the Rightmost Mass Spectrum Peak Reflect a Molecule’s Relative Molecular Mass, Irrespective of Its Intensity?

  Mass spectrometry (MS) is a technique employed to determine the mass, structure, and composition of molecules. In a mass spectrum, the horizontal axis represents the mass-to-charge ratio (m/z), while the vertical axis represents signal intensity. Each peak corresponds to a specific ion or molecular fragment, with the peak intensity reflecting its relative abundance.   The rightmost peak in a mass spectrum is generally identified as the molecular ion peak (M⁺), which corresponds to the intact ion of th......

• • How Can Relative Abundance Be Calculated?

  In mass spectrometry, relative abundance typically refers to the natural abundance of isotopes-that is, the proportion of a given isotope present in a sample. This parameter can be measured using a mass spectrometer. The general procedure for calculating relative abundance is as follows:   1. Sample Collection Collect samples that contain the isotopes of interest.   2. Ionization Convert the molecules in the sample into ions using the ion source of the mass spectrometer.   3. Mass Spectrometric Analys......

• • How Can the Differences Between Protein and mRNA Levels Be Explained?

  Differences between protein levels and mRNA levels are a well-documented phenomenon in biological research, arising from regulation at multiple stages of gene expression. Key factors contributing to these differences include:   1. Post-Transcriptional Regulation A variety of regulatory mechanisms operate between mRNA synthesis (transcription) and protein synthesis (translation). Factors such as mRNA stability, transport, and subcellular localization determine the pool of mRNA available for translation......

• • How Can the Isoelectric Point of a Peptide Be Calculated?

  The process to calculate the isoelectric point of a peptide generally involves the following steps:   1. Determine the Amino Acid Composition of the Peptide First, the amino acids that make up the peptide must be identified. Each amino acid has distinct acid-base properties, which contribute to the peptide's isoelectric point.   2. Determine the pKa Values of Each Amino Acid Each amino acid has a specific pKa value that indicates the ionization state of its carboxyl and amino groups under acidic condi......

• • What Types of Substances Can Be Measured Using Liquid Chromatography?

  Liquid Chromatography (LC) is an analytical technique used for the separation and analysis of complex mixtures. It is capable of detecting and quantifying a wide range of substances, including but not limited to:   1. Organic Compounds Such as fatty acids, alcohols, phenols, ketones, aldehydes, esters, organic acids, and organic bases.   2. Biological Macromolecules Including proteins, peptides, nucleic acids (DNA and RNA), and polysaccharides.   3. Pharmaceutical Compounds and Their Metabolites Such ......

• • How Can the Internal Standard Method Quantify Compounds in LC-MS Without a Reference Standard?

  In liquid chromatography-mass spectrometry (LC-MS) analysis, when a reference standard for the target compound is unavailable, quantification can be performed using the internal standard method. This approach involves introducing an internal standard (IS), a compound with similar chemical properties but slight structural or isotopic differences from the target analyte, into the sample. The concentration of the target compound is then determined based on the response ratio between the internal standard......

• • How Can the Relative Molecular Mass of a Protein Be Determined?

  The relative molecular mass of a protein (also referred to as molecular weight) can be determined using several analytical techniques:   1. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) SDS-PAGE is a widely used electrophoretic method for estimating protein molecular mass. During electrophoresis, proteins migrate as bands toward the anode, with their migration rate inversely proportional to their molecular weight. By co-electrophoresing the sample with molecular weight markers a......

• • What Are the Key Differences Between N-Linked and O-Linked Glycosylation?

  N-linked and O-linked glycosylation are the two predominant forms of glycosylation in glycoproteins, both playing essential roles in various biological processes. Their key differences are summarized as follows:   N-Linked Glycosylation 1. This type of glycosylation occurs on the amide nitrogen of asparagine (Asn) residues.   2. It requires a specific consensus sequence, Asn-X-Ser/Thr, where X can be any amino acid except proline.   3. N-linked glycosylation is initiated in the endoplasmic reticulum (......

• • Why Is a Secondary Antibody Necessary for Autoradiographic Detection in Western Blotting?

  Principle of the Western Blotting Assay Western blotting involves separating the target protein and transferring it onto a membrane. The target protein on the membrane is first recognized by a specific primary antibody. The secondary antibody then binds to the primary antibody at its binding site on the target protein. Finally, the secondary antibody, conjugated with an enzyme or a radioactive isotope, generates a detectable signal that allows for the identification of the target protein.   Why Are Bo......