Protein Analysis FAQ

• • How to Manually Integrate Peak Area on HPLC

  High-performance liquid chromatography (HPLC) is usually equipped with data processing software for real-time or offline chromatographic data analysis. In the data processing software, peak area can be integrated automatically or manually. Below are the general steps for manually integrating peak area in HPLC data processing software: 1. Open Chromatographic Data Open the chromatographic data file that needs manual integration in the data processing software. 2. Select Manual Integration Mode In the........

• • How to Calculate Protein Expression Levels in Proteomics

  In proteomics research, common methods to measure protein expression levels are based on relative or absolute quantification using mass spectrometry data. Common quantification methods include labeling and label-free approaches: 1. Labeling Quantification Proteins or peptides are labeled using isotope tagging techniques, such as SILAC, ICAT, TMT, and iTRAQ. The relative change in protein expression is calculated by comparing the signal intensity of isotope labels in different samples. Log2-fold change......

• • What Are the Forms of Protein Modifications and How Do They Affect Cell Signaling

  Types of Protein Modifications 1. Phosphorylation and Dephosphorylation Phosphorylation involves adding or removing phosphate groups to specific amino acid residues (usually serine, threonine, or tyrosine) on proteins. This is the most common form of protein modification, affecting protein structure and function, playing a key role in many signaling pathways. 2. Ubiquitination Ubiquitin proteins are linked to the lysine residues of target proteins through a system of three enzymes.

• • Protein Analysis: Can Protein WB Expression Be Verified by qPCR

  Western Blot (WB) expression of proteins and qPCR (quantitative polymerase chain reaction) actually analyze different levels of biological information. WB is mainly used to detect protein expression levels, while qPCR is used to detect mRNA expression levels of genes. Although there is some correlation between them, they measure different biomolecules. Western Blot (WB) is an immunoassay technique used for qualitative and quantitative analysis of target protein expression levels in cells or tissues.

• • Why Does the Protein Purification Process Always Fail to Bind to the Column

  During protein purification, if the protein is not effectively binding to the column, there could be several reasons: 1. Inappropriate Buffer pH Protein binding usually depends on its isoelectric point and the pH of the buffer. If the pH is not suitable, the protein may not bind correctly. 2. Salt Concentration Too High or Too Low Salt concentration affects protein solubility and binding ability. If the salt concentration is not appropriate, it may hinder protein adsorption. 3. Column Issues The column.....

• • GC-MS and LC-MS Sample Requirements: Can Cell Fermentation Broth Be Used

  GC-MS and LC-MS are commonly used analytical techniques for identifying and quantifying compounds in complex samples. However, due to differences in their working principles and application fields, their sample requirements vary. GC-MS Sample Requirements 1. Volatility Samples for GC-MS analysis must be volatile, as they need to be distilled into the gas column at high temperatures. 2. Stability Samples must be stable at the high temperatures in GC, without decomposition. 3. Purity As with LC-MS, minimize..

• • How to Identify the Name of a Compound/Substance by Analyzing a Mass Spectrum

  A mass spectrum is a graphical representation where the x-axis represents the mass-to-charge ratio (m/z) and the y-axis represents relative abundance. Each peak represents an ion, with its position indicating the ion's m/z value and its height indicating the ion's relative abundance. 1. Identifying the Molecular Ion Peak The molecular ion peak (M+ peak) is typically the highest peak in the mass spectrum, representing the intact molecular ion. This peak provides information about the compound's molecular....

• • 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.....