Protein Analysis FAQ

• • 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.....

• • Methods for Isolating and Extracting Complete Bacterial Cell Walls

  Extracting a complete bacterial cell wall is a challenging task, as it requires removing all internal components while minimizing damage to the cell wall. Methods for extracting intact cell walls can be broadly categorized based on the lysis technique, including alkaline hydrolysis, enzymatic digestion, chemical methods, ultrasound, and mechanical methods. 1. Alkaline Hydrolysis Bacterial cell walls are placed in an alkaline solution (e.g., sodium hydroxide or potassium hydroxide) to hydrolyze proteins.....

• • Can IP Extraction Proteins Be Stored at -80°C Like WB Proteins

  Yes, protein samples for immunoprecipitation (IP) can usually be stored at -80°C, just like protein samples for Western blot (WB). The key is to avoid repeated freeze-thaw cycles during storage, as this may cause protein degradation or inactivation. When using the sample, it should be thawed completely and used in one go.

• • Q&A of Protein Glycosylation Analysis

  This FAQ guide provides clear answers to common questions on protein glycosylation analysis. It covers key methods including chromatography, lectin binding assays, enzymatic digestion, and glycan labeling, along with strategies for distinguishing N-linked and O-linked glycosylation, deglycosylation techniques, sample preparation best practices, and troubleshooting tips to optimize glycosylation profiling by mass spectrometry.