Protein Analysis FAQ

• • When Performing a WB Experiment, Why Is It Necessary to Add Proteases and Inhibitors When Extracting Proteins

  During a Western Blot (WB) experiment, the addition of proteases and inhibitors during the protein extraction process is necessary for the following reasons: Addition of Proteases 1. The presence of proteases may degrade the target proteins; therefore, adding proteases during protein extraction can prevent protein degradation. 2. Proteases can degrade intracellular proteins, including receptor proteins on the cell membrane, cytoskeletal proteins, etc. By adding proteases, these intracellular proteins can...

• • Will the Protein Sample Degrade if Left at Room Temperature Overnight

  Protein stability is primarily influenced by the specific protein, sample composition, and storage conditions. However, for most proteins, storage at room temperature overnight can result in degradation. For critical protein samples, freezing immediately following purification and storing at -80°C is advisable. Alternatively, adding suitable stabilizers or protease inhibitors and storing at 4°C for short-term preservation can help maintain stability. For long-term storage, samples should be kept at -20°C...

• • Why Does Loading Too Large a Sample Volume in Gel Filtration Chromatography Lead to Peak Overlapping

  Gel filtration chromatography (also referred to as gel permeation chromatography or size-exclusion chromatography) is a widely used biochemical technique for separating molecules based on size. When an excessively large sample volume is loaded, peak overlapping may occur. This phenomenon can be attributed to the following factors: 1. Sample Distribution In gel filtration chromatography, molecules are separated according to their hydrodynamic size as they pass through the gel matrix. Larger molecules are....

• • What Software Is Used for Protein Mass Spectrometry? Why Is Protein Identification Yield Low

  In protein mass spectrometry analysis, a variety of software tools are available for qualitative protein identification. Below are several commonly used platforms: 1. Mascot A widely adopted commercial tool developed by Matrix Science. It supports data interpretation from various mass spectrometry techniques by performing database searches across multiple protein repositories. 2. SEQUEST Originally developed by John Yates III and currently distributed by Thermo Fisher Scientific, SEQUEST is typically ......

• • How to Analyze Circular Dichroism Data to Determine the Proportions of Different Secondary Structures

  Circular Dichroism (CD) spectroscopy is a widely used technique for investigating the secondary structures of proteins and other biomacromolecules. It operates on the principle that such molecules exhibit differential absorption of left- and right-handed circularly polarized light at specific wavelengths. The following are some fundamental steps and approaches for analyzing CD data, which may serve as a useful guide: 1. Baseline Correction Begin by performing baseline correction to remove instrumental .....

• • Which Proteins Should You Run in WB to Test Antibody Effects on Mouse Fatty Liver

  To evaluate whether hepatic steatosis in mice is alleviated following treatment with a specific antibody, the expression levels of the following proteins associated with hepatic steatosis can be assessed by Western blot (WB): 1. Proteins Related to Lipid Metabolism Hepatic steatosis is characterized by dysregulated lipid metabolism. Therefore, it is relevant to detect proteins involved in lipid metabolic processes, such as Fatty Acid Synthase (FAS), enzymes involved in fatty acid oxidation (e.g., CPT1......

• • How to Determine the Purity of the Target Protein Using Electrophoresis Results

  Electrophoresis results provide information regarding the molecular weight and charge of proteins, but they do not directly quantify protein purity. Nonetheless, it is possible to assess protein purity indirectly through electrophoretic analysis. Following electrophoresis, the gel must be stained to visualize the protein bands. By analyzing the electrophoretic image, an initial assessment of the target protein’s purity can be made. If a single, distinct band is observed at the expected molecular weight.....

• • How to Locate Disulfide Bonds

  Identifying the positions of disulfide bonds in proteins is a complex process that typically involves the integration of multiple experimental and computational approaches. The following are commonly used strategies for disulfide bond localization: 1. X-ray Crystallography This is the most widely applied technique for resolving the three-dimensional structure of proteins, including the identification of disulfide bonds. When exposed to X-rays, protein crystals produce diffraction patterns that can be ......

• • How to Purify Microtubule Proteins

  Microtubule proteins, primarily composed of α- and β-tubulin subunits, are essential structural components of the cytoskeleton. Their purification involves a multistep process, as outlined below: 1. Cell Collection A suitable cell line is selected and harvested by centrifugation to pellet the cells, thereby removing the culture medium. 2. Cell Lysis The cell pellet is resuspended in a microtubule-stabilizing buffer (e.g., PEM buffer containing 100 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, pH 6.9) supplemented with..

• • Does a Disulfide Bond Belong to the Primary Structure of a Protein

  To elucidate this issue, we provide a comprehensive explanation from two perspectives: the definition of protein structure and the characteristics of disulfide bonds. Definition of Protein Structure 1. Primary Structure The primary structure of a protein refers to its specific sequence of amino acids. This represents the most fundamental level of protein structure and determines all subsequent structural levels. In the primary structure, amino acids are covalently linked by peptide bonds, which are formed..