Protein Analysis FAQ

• • How to Extract Small Intestine Proteins and Which Part to Use for ZO-1 Expression Analysis

  Protein Extraction 1. Sample Preparation Excise a designated section of the small intestine from the experimental animals and immediately immerse it in ice-cold physiological saline to remove residual luminal contents. 2. Tissue Homogenization (1) Homogenize the intestinal tissue using a mechanical homogenizer. During this process, the tissue is physically disrupted to release intracellular proteins into the extraction buffer. (2) Use an appropriate buffer, such as phosphate-buffered saline (PBS) ..........

• • What Are the Basic Strategies of Proteomics Research

  The basic strategies of proteomics research typically include the following aspects: 1. Sample Preparation and Protein Extraction Initially, proteins are extracted from the biological samples under investigation—such as cells, tissues, or whole organisms—and subsequently subjected to necessary purification and processing steps to facilitate downstream analyses. 2. Protein Separation To separate the extracted proteins, various analytical techniques are employed, including two-dimensional gel.........

• • What Will Happen if the Voltage Is Too Low During the Membrane Transfer Process in WB

  1. Reduced Transfer Efficiency Voltage serves as the driving force facilitating the movement of membrane material from one surface to another. When the voltage is insufficient, it may fail to adequately support this process, leading to decreased transfer efficiency. 2. Non-uniform Transfer Insufficient voltage can hinder the even distribution of membrane material, potentially resulting in inconsistencies such as variable thickness, surface irregularities, or structural imperfections in the transferred......

• • How to Quantitatively Analyze the Results of Immunohistochemistry and Western Blot

  Quantitative Analysis of Immunohistochemistry 1. Image Acquisition Use a microscope to capture images of stained tissue sections, ensuring that all imaging parameters (e.g., exposure time, light source intensity) are maintained consistently across all samples. 2. Image Analysis Open the acquired images using image analysis software such as ImageJ. 3. Threshold Setting Set a threshold based on background levels and staining intensity to accurately differentiate stained regions from the background.

• • How to Analyze Mass Spectrometry Results and Find Interacting Proteins

  Mass spectrometry analysis typically involves three key steps: data preprocessing, protein identification, and quantitative analysis. Data Preprocessing 1. Mass calibration: Mass spectra are calibrated using known reference standards to ensure the accuracy of m/z measurements. 2. Peak detection: Ion peaks in the spectra are identified and integrated to facilitate subsequent protein or peptide identification. Protein Identification 1. Database searching: Software tools such as Mascot, SEQUEST, or MaxQuant...

• • Can Ultraviolet Spectrophotometry Be Used for the Quantitative Analysis of Amino Acids and Proteins

  Ultraviolet spectrophotometry (UV-Vis spectrophotometry) is a widely employed technique for the quantitative analysis of amino acids and proteins. 1. Quantitative Analysis of Amino Acids Certain amino acids, such as tryptophan and tyrosine, possess intrinsic ultraviolet absorbance, particularly around 280 nm. Therefore, UV-Vis spectrophotometry can be used directly for their quantitative determination. In contrast, amino acids that lack UV absorbance require chemical derivatization or alternative ..........

• • Why Does Purified Protein Show 60kDa Instead of the Expected 107kDa During SDS-PAGE

  If a purified protein displays a significantly lower apparent molecular weight on SDS-PAGE than expected (e.g., expected 107 kDa but observed as 60 kDa), several potential explanations should be considered: 1. Protein Degradation The most common explanation is partial degradation of the protein during extraction or purification, often caused by endogenous protease activity. To prevent this, protease inhibitors should be added during protein extraction and purification, and all procedures should be carried..

• • Why Do Two Bands Appear in SDS-PAGE After Co-IP to Detect Protein-Protein Interaction

  Co-immunoprecipitation (Co-IP) is a widely used method for detecting protein-protein interactions. In brief, when researchers hypothesize that two proteins may interact, they can use an antibody specific to one of the proteins to immunoprecipitate it along with any proteins bound to it. The precipitated protein complex is then subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), followed by analysis through Western blotting. The appearance of two bands on the SDS-PAGE gel......

• • Is It Necessary for Proteins Used in Co-Immunoprecipitation to Have Tags Like Flag or HA

  In co-immunoprecipitation (Co-IP) experiments, it is typically necessary to use specific antibodies to recognize and isolate the target protein. These antibodies bind to the target protein and allow for its co-precipitation along with associated interacting proteins. In some cases, to improve the efficiency and specificity of Co-IP, the target protein may need to be engineered to carry specific epitope tags, such as the FLAG tag or HA tag. These epitope tags facilitate the isolation of the target protein...

• • Why Can Polyacrylamide Gel Electrophoresis Separate More Protein Bands

  Polyacrylamide gel electrophoresis (PAGE) is a widely used biochemical technique that enables the separation of proteins primarily based on their molecular weight or charge. The following factors contribute to the ability of PAGE to separate a greater number of protein bands: 1. Molecular Sieving Effect The pore size of polyacrylamide gels can be precisely controlled by adjusting the gel concentration. High concentrations create smaller pores suitable for resolving proteins with low molecular weight........