Protein Analysis FAQ

• • 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......

• • How Are LC-MS Parameters Typically Configured for Protein Analysis?

  In liquid chromatography-mass spectrometry (LC-MS) analysis of proteins, parameter selection depends on the characteristics of the sample and the analytical objectives. The typical LC-MS parameters are outlined as follows:   Chromatographic Conditions 1. Column Type C18 reversed-phase column (15–25 cm length, 2.1 mm inner diameter, 3–5 µm particle size).   2. Mobile Phases (1) Phase A: 0.1% formic acid or trifluoroacetic acid in water. (2) Phase B: 0.1% formic acid or trifluoroacetic acid in acetonitr......

• • Why Are Protein Bands Not Visible After Destaining in SDS-PAGE?

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used method for protein separation based on molecular weight. If no bands are observed after the destaining step, several factors may be responsible:   1. Inadequate Sample Preparation Insufficient protein concentration or improper sample handling may result in undetectable bands. It is crucial to ensure that the protein concentration is adequate and that proper sample preparation steps, such as heat denaturation and the ......

• • What Are the Differences Between AKTA Fast Protein Liquid Chromatography and HPLC in Protein Purification?

  Both AKTA and HPLC are chromatographic techniques commonly used for the purification of proteins and other biomacromolecules. However, they differ significantly in terms of design, applications, and operational characteristics:   Primary Applications 1. AKTA The AKTA system, developed by GE Healthcare (now Cytiva), is specifically designed for the purification of biomolecules, particularly proteins. It is widely used in affinity chromatography, ion-exchange chromatography, and size-exclusion chromatog......

• • What Does 'Pan-Cancer' Mean in Pan-Cancer Analysis?

  The term "pan-cancer" is a relatively recent concept frequently used in cancer genomics and transcriptomics research. It refers to an integrative analysis spanning multiple cancer types or subtypes, rather than focusing on a single cancer type. The primary goal of pan-cancer analysis is to identify shared genes, pathways, biomarkers, or other molecular features across diverse cancer types, potentially governed by common molecular mechanisms or biological processes.   One of the most prominent initiati......