Protein Analysis FAQ

• • What Are the Analytical Methods for Molecular Weight Determination

  Molecular weight (MW) detection is a commonly used analytical technique in chemical, biochemical, and molecular biology research. Depending on the properties of the substance being studied and the problem to be solved, several different analytical methods can be employed: 1. Mass Spectrometry (MS) Mass spectrometry is a widely used method for molecular weight determination. It works by measuring the mass-to-charge ratio (m/z) of sample molecules to determine their molecular weight. The process involves.....

• • Principles of UV Absorption in Proteins and Nucleic Acids: Differences and Charge Mechanism

  Principles of UV Absorption Both proteins and nucleic acids can absorb ultraviolet (UV) light. This absorption is due to the transition of certain π-electrons and unpaired n-electrons in their molecules to higher energy states after absorbing light of a certain energy. The aromatic amino acids in proteins (such as tyrosine, phenylalanine, and histidine) are mainly responsible for UV absorption. The aromatic rings in these amino acids contain π-electrons, which can transition to a higher energy state upon...

• • What Is a Trap Column in LC-MS/MS Analysis and What Role Does It Play

  In LC-MS/MS analysis, a trap column is a small fixed-phase column typically used for sample pre-treatment before the actual analysis. The main functions of a trap column are as follows: 1. Concentration and Enrichment The trap column can concentrate diluted samples to enhance the detection sensitivity of target substances. By temporarily capturing target compounds in the trap column, impurities can be effectively removed, and the target substances can be enriched on the column. 2. Removal of Impurities.....

• • How to Interpret Proteomics Mass Spectrometry Data

  Proteomics mass spectrometry data are obtained using a mass spectrometer to analyze protein composition and structure. Observing and interpreting mass spectra requires understanding the basic components and features of the spectra. Basic Features of Mass Spectra 1. X-axis Represents m/z (mass-to-charge ratio). 2. Y-axis Represents intensity (usually relative intensity, not directly comparable between different spectra). Common Types of Mass Spectra 1. MS1 (Primary Mass Spectrum) Displays the m/z and........

• • Principles and Steps of Western Blotting, Differences with ELISA, and Choosing the Right Method for Protein Quantification

  Western Blotting 1. Experimental Principle Western Blotting is a technique used to detect the presence and expression levels of specific proteins in samples. The basic principle involves separating proteins using SDS-PAGE electrophoresis, transferring the proteins to a membrane (e.g., PVDF), and then detecting the target protein using specific antibodies. 2. Experimental Steps (1) Protein Sample Preparation: Extract proteins from cells or tissues. (2) SDS-PAGE Electrophoresis: Use electrophoresis to........

• • How to Perform Native Protein Electrophoresis

  Native Polyacrylamide Gel Electrophoresis (Native PAGE) is a protein separation technique performed under non-denaturing conditions. The goal of Native PAGE is to separate proteins based on their charge, shape, and size while preserving their native structure and biological activity. The operation is relatively simple, and the steps are as follows: 1. Prepare Polyacrylamide Gel Choose the appropriate concentration of polyacrylamide gel, typically between 5-20%. Higher concentrations separate smaller........

• • How to Choose the Optimal Gel Concentration and Buffer pH for Protein SDS-PAGE and Molecular Weight Determination

  Gel Concentration Selection 1. The choice of gel concentration should be based on the molecular weight range of the protein to be tested. Generally, lower gel concentrations are suitable for larger proteins, while higher concentrations are better for smaller proteins. 2. Commonly used gel concentrations range from 8% to 15%. For larger proteins, a lower concentration such as 8% is recommended, while for smaller proteins, higher concentrations like 12% or 15% are ideal. 3. If the molecular weight of the.....

• • Protein Precipitation After Heating Denaturation in WB Experiments

  In Western Blot (WB) experiments, protein precipitation after heating denaturation is a common issue. This is usually caused by the following factors: 1. High Protein Concentration If the protein concentration in the sample is too high, excessive aggregation and precipitation may occur during heating. 2. Inappropriate Sample Buffer The sample buffer used may not be suitable for the protein being analyzed. For example, improper pH or salt concentration of the buffer can affect protein solubility.

• • How to Write a COG Functional Annotation Analysis: Is It Just About Reporting the Most Abundant Category

  Using the COG database for functional annotation analysis not only reveals the most abundant functional categories but also provides a detailed distribution of genes across various biological categories. Below is a method for writing a COG functional annotation analysis, which we hope will be helpful to you. 1. Introduction In the introduction, briefly introduce the significance of the COG database and its main uses. Explain the reasons for using the COG database to functionally annotate a specific genome..

• • How to Select Quantitative & Qualitative Ion Pairs in LC-MS and Enhance Ionization Efficiency with Mobile Phase Additives

  When developing quantitative methods in LC-MS, determining the quantitative and qualitative ion pairs is critical. Below are some guidelines on how to choose them: 1. Quantitative Ion Pairs Typically, the ions with the highest intensity are selected as quantitative ions because they offer the best signal-to-noise ratio. In practice, the full-scan mass spectrum of the target compound should first be obtained, and the highest abundance, representative ions should be identified. These ions can be molecular....