Protein Analysis FAQ

• • Common Types of Proteins Used in WB Experimental Protein Extraction

  When performing protein extraction for WB experiments, the commonly extracted protein fractions include the following: Total Cellular Proteins This is the most commonly extracted protein fraction, obtained by lysing both the plasma and nuclear membranes to release all intracellular proteins. This extraction approach is widely used to analyze overall protein expression in cells. Nuclear Proteins Nuclear proteins are isolated by selectively lysing the cytoplasmic membrane while preserving nuclear integrity...

• • What Are the Methods for Determining the Molecular Weight Distribution of Polymers

  Molecular weight distribution (MWD) of polymers describes the relative proportions of polymer molecules with different molecular weights within a given sample. Determining MWD is crucial for understanding the structure and properties of polymeric materials. The following are some commonly used methods for analyzing the molecular weight distribution of polymers: 1. Gel Permeation Chromatography (GPC/SEC) Gel permeation chromatography (also known as size-exclusion chromatography, SEC) is the most widely......

• • What Should Be Done if the Flow Rate Is Too Slow During Protein Purification Through a Nickel Column

  Nickel columns (Ni-NTA) are a widely used method for protein purification, utilizing the binding of His-tagged proteins to six consecutive histidine residues (His-tag). If you experience a slow flow rate while purifying proteins using a nickel column, you can try the following approaches: Check the Piston and Connections Ensure that the piston and connections are correctly installed and undamaged. If any issues are found, replace the piston or connections as needed. Reduce the Sample Concentration .........

• • What Determines the Migration Direction of Proteins in an Electric Field

  In different types of electrophoresis, the migration direction of proteins is determined by their charge state as well as the electrophoretic conditions and medium used. Below are several common types of electrophoresis and the factors determining protein migration direction: 1. Native PAGE (Non-denaturing Electrophoresis) In native PAGE, proteins maintain their natural state and are not denatured. The migration direction of proteins depends on their charge state at the pH of the electrophoresis buffer.....

• • Is the Sample Preparation for Protein Detection Using MALDI-TOF and QE Mass Spectrometry the Same

  MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and QE (Quadrupole-Orbitrap) mass spectrometry are two different mass spectrometry techniques, and their pretreatment steps in protein detection have some differences. The pretreatment for MALDI-TOF tends to involve directly mixing the sample with a matrix to form crystals, whereas the QE mass spectrometry pretreatment focuses more on liquid chromatography separation and ionization of proteins or peptides.

• • Why Are There So Many Bubbles on the Edge of the Western Blot Gel

  The formation of bubbles along the edges of Western blot gels can result from several factors, including the following: 1. Bubbles Introduced During Gel Preparation During the preparation of polyacrylamide gels, inadequate mixing or uneven stirring of the gel solution can lead to bubble formation. To minimize this, the gel solution should be thoroughly mixed and then allowed to stand for a period before use, facilitating the escape of trapped air. 2. Bubbles Formed During Gel Pouring If the gel solution....

• • How to Use Bioinformatics to Screen Inhibitors for Target Proteins

  Screening inhibitors for target proteins using bioinformatics is a key step in the drug discovery process, commonly referred to as virtual screening or computational screening. The following are the basic steps for screening target protein inhibitors based on bioinformatics: Target Protein Structure Acquisition Obtain the three-dimensional structure of the target protein from PDB (Protein Data Bank) or other relevant databases. If no experimental structure is available, consider using homology modeling.....

• • The Main Software Used for Predicting Post-Translational Modifications of Protein Sequences

  The prediction of post-translational modifications (PTMs) in protein sequences is of great importance in bioinformatics and systems biology, as PTMs play a crucial role in regulating protein function, localization, and interactions. Several computational tools have been developed to predict PTMs, with some of the most commonly used ones listed below: 1. PhosphoSitePlus A comprehensive database providing information on experimentally identified post-translational modifications, particularly in disease-......

• • Can I Reboil My WB Sample After Storing It in the Fridge

  Yes. If the sample has undergone prior boiling and has been stored at 4°C, it is recommended to reheat it before use to ensure that the components in the loading buffer, such as SDS and DTT (or β-mercaptoethanol), function effectively, allowing complete protein denaturation. Before reheating, ensure that the sample is fully thawed and gently mixed to prevent phase separation or precipitation of its components, which could affect experimental results.

• • Western Blot: Why Did This Situation Occur Where the Protein Ran onto the Marker

  When proteins run onto the marker during a Western blot experiment, several factors may contribute to this phenomenon: 1. Excessive Sample Loading Loading an excessive amount of sample can cause proteins to migrate beyond the resolving capacity of the gel, resulting in some proteins running onto the marker. This typically occurs when the protein concentration is too high or when the sample volume is not accurately controlled during loading. Solution: Determine the loading volume based on both the protein...