Protein Analysis FAQ

• • Why Post-Translational Modifications of Intracellular Proteins Are Necessary and What Are the Types

  Post-translational modifications (PTMs) of proteins in the cell are crucial because these modifications not only ensure that proteins have the proper conformation and function, but also regulate their localization and stability within the cell. PTMs provide a fine mechanism for regulating protein functions and activities, and are involved in controlling various biological processes. Why Proteins Need Post-Translational Modifications 1. Functional Diversity Modifications increase the functional diversity....

• • Is the IOD Value Being Measured? Using ImageJ to Analyze Grayscale Values and Calculate Protein Purity Percentage

  The IOD (Integrated Optical Density) value, or integrated optical density, is the integral of the optical density within a specific region, generally used to analyze the color or grayscale distribution in an image. In SDS-PAGE protein purity analysis, it can be used to measure the intensity of specific protein bands, and this intensity is typically proportional to the protein content. In SDS-PAGE analysis, we generally do not directly measure the IOD value. Instead, we assess the relative abundance of......

• • What Causes Bromophenol Blue Diffusion in the Concentrating Gel During WB Protein Electrophoresis

  Bromophenol blue is a commonly used tracking dye in SDS-PAGE electrophoresis to monitor the migration of samples. Its migration speed is generally similar to that of small molecular weight proteins, making it useful for observing the electrophoresis process. Causes of Diffusion Phenomenon 1. Gel Concentration If the concentration of the concentration gel is improperly set, it may lead to the diffusion of bromophenol blue. 2. Electrophoresis Conditions Voltage, current, buffer pH, and composition can all....

• • How to Isolate and Purify Protein Bands After SDS-PAGE

  After SDS-PAGE, if you want to recover a specific protein band from the gel, you can purify it using the following steps: 1. Staining and Destaining the Gel First, stain the entire gel with an appropriate dye (such as Coomassie Brilliant Blue). After a set time, destain the gel until the desired protein band is clearly visible. 2. Cutting the Gel Using a clean, sharp blade or scissors, cut the gel along the stained protein band, minimizing the non-protein areas around the band. 3. Protein Extraction........

• • SDS-PAGE Concentration Calculation Method

  When preparing SDS-PAGE gels, the concentration of the gel refers to the total concentration of acrylamide, which determines the pore size of the gel and affects the protein separation efficiency. The higher the concentration, the smaller the pores, suitable for separating low molecular weight proteins; the lower the concentration, the larger the pores, suitable for separating high molecular weight proteins. Formula for Calculating Gel Concentration Total gel concentration (T) = acrylamide concentration....

• • What Are the Differences in Results When Dissolving Samples in 50% Methanol vs 100% Methanol for HPLC Measurements

  In high-performance liquid chromatography (HPLC) experiments, using different concentrations of solvents (such as 50% methanol and 100% methanol) to dissolve samples may have the following impacts on the results: 1. Solubility Different concentrations of methanol may affect the solubility of the sample. If the sample does not dissolve well in 50% methanol, it may cause partial precipitation, resulting in incomplete detection of all components during HPLC analysis.

• • Why SDS-PAGE Can Be Used for Protein Purification

  SDS-PAGE electrophoresis is a commonly used technique for protein separation and purification. Its principle relies on the properties of SDS (sodium dodecyl sulfate) and polyacrylamide gel to separate protein samples into different-sized bands, thus achieving protein purification. 1. Role of SDS SDS is a surfactant that interacts with hydrogen bonds and hydrophobic interactions within protein molecules, unfolding them into linear structures. SDS also imparts a negative charge to proteins, causing them to...

• • Types of Interactions Between Proteins and Small Molecules

  Interactions between proteins and small molecules are crucial, as they often involve biological processes such as drug mechanisms, enzyme substrate recognition, and signal transduction. These interactions can be classified based on the types of forces involved, including: 1. Electrostatic Interactions (Charge-Charge Interactions) These interactions arise from the attraction between opposite charges (e.g., between cations and anions) or repulsion between like charges. They are often significant in protein...

• • How to Estimate Protein Concentration Based on SDS-PAGE Gel

  SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a commonly used protein electrophoresis technique, typically used to analyze protein molecular weight and purity. To estimate protein concentration based on SDS-PAGE gel, follow these steps: 1. Create a Standard Curve Run a gel containing proteins with known concentrations to establish a relationship between protein concentration and the intensity (grayscale value) of the bands on the gel. This standard curve is usually generated us....

• • How to Verify if a Membrane Protein is Expressed on the Cell Membrane

  Verifying whether a membrane protein is expressed on the cell membrane can be done through various experimental methods, such as: 1. Fluorescence Microscopy Fuse the membrane protein with a fluorescent protein (e.g., GFP) and transfect it into the target cells. Observe the cells using a fluorescence microscope to check if the fluorescence signal is concentrated in the cell membrane region. 2. Biochemical Separation Use methods like centrifugation to separate the cytoplasmic and membrane components from.....