Protein Analysis FAQ

• • How to Interpret the Western Blot for Protein Phosphorylation

  Western blot (WB) is a widely used technique for analyzing protein expression levels and post-translational modifications. When using phospho-specific antibodies to detect protein phosphorylation, the results can be interpreted from the following perspectives: 1. Check Background Clarity Begin by ensuring that the membrane exhibits a low background signal, allowing bands to be clearly distinguished. Excessive background may result from non-specific binding or insufficient blocking.

• • Why Is There a Step in Western Blot That Requires Determining the Protein Concentration in the Tissue

  Determining the protein concentration of tissue samples serves the following key purposes: 1. To Quantify the Relative Protein Content of Samples Measuring the protein concentration allows for accurate assessment of the relative protein content across samples. In Western blot experiments, samples from different groups—such as control and experimental—are often compared. Knowing the protein concentration ensures that equal amounts of protein are loaded into the gel, allowing for valid comparisons during.....

• • WB Shows 20kDa Increase, How to Use Dephosphorylation and Deglycosylation to Confirm Post-Translational Modifications

  In Western blot (WB) experiments, an observed increase of approximately 20 kDa in the molecular weight of a target protein compared to its predicted mass may suggest the occurrence of post-translational modifications. To verify this possibility, the following steps can be taken: Dephosphorylation Verification 1. Sample Preparation Ensure that a sufficient amount of protein sample is available for analysis. 2. Selection of an Appropriate Phosphatase Alkaline phosphatases, such as Lambda protein phosphatase..

• • Why Are There So Many Keratins in My Mass Spectrometry Results

  The frequent detection of keratins in mass spectrometry results is often attributed to sample contamination during experimental procedures. Keratins are a group of abundant and stable structural proteins commonly found in the epidermis, hair, nails, and other tissues of humans and animals. Owing to their high abundance and resilience, keratins can be readily introduced into samples throughout the experimental workflow, resulting in their pervasive presence in mass spectrometry data. The primary sources.....

• • Can WB Without SDS Work for Small Proteins (10KD)? Is Tris Used Instead for Gel Balance

  Small proteins (10 kDa) are typically analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in Western Blot (WB) experiments. SDS, as a surfactant, denatures proteins and imparts a uniform negative charge, enabling their separation based solely on molecular weight. In the absence of SDS during electrophoresis, proteins are not fully denatured. As a result, their migration behavior is influenced not only by molecular size but also by their native conformation and intrinsic charge, leading to......

• • 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...