Protein Analysis FAQ

• • Why Does the Target Protein Show a Double Band in the Input but a Single Band in the IP?

  In immunoprecipitation (IP) experiments, observing a double band for the target protein in the input sample but only a single band in the IP sample may be attributed to several factors:   Protein Modifications The target protein in the input sample may exist in different post-translationally modified forms (e.g., phosphorylation, glycosylation), leading to variations in electrophoretic mobility and resulting in a double band. In the IP sample, the antibody might specifically recognize and precipitate ......

• • Does a Fold Change Threshold of 1.5 Enhance Acceptance in Proteomics Differential Protein Screening?

  In proteomics, selecting a fold change (FC) threshold of 1.5 is commonly used to identify differentially expressed proteins. However, the optimal FC threshold can vary depending on the specific study design, sample size, and research objectives. For instance, studies with smaller sample sizes may require more stringent thresholds to ensure reliability, while larger studies might adopt more lenient criteria to detect a broader range of differential proteins.   Additionally, combining FC thresholds with......

• • What Distinguishes Anti-HLA-DR Antibodies from Broad-Spectrum Anti-MHC Class II Antibodies?

  Anti-HLA-DR antibodies and anti-MHC class II antibodies are both directed against MHC class II molecules but differ in terms of species specificity and molecular targets. Although they share similar immunological functions, their primary distinction lies in the scope of application and recognition specificity:   Anti-HLA-DR Antibodies HLA-DR is a subtype of human MHC class II molecules, where HLA (Human Leukocyte Antigen) denotes the human major histocompatibility complex. Anti-HLA-DR antibodies speci......

• • Which Software Is Best Suited for Processing Shimadzu Data?

  The optimal software for processing Shimadzu data depends on the specific analytical instrument and application area. Shimadzu offers a variety of analytical instruments, such as High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Mass Spectrometry (MS), each potentially supported by different software.   Common Shimadzu Data Processing Software 1. LabSolutions Shimadzu's primary data processing software, widely used for instruments like HPLC and GC. LabSolutions provides data ......

• • How Can Proteins with Similar Structures and Closely Related Molecular Weights Be Effectively Separated?

  For proteins that exhibit similar structural characteristics and closely related molecular weights, conventional separation techniques may be insufficient to achieve effective resolution. Nevertheless, several strategies can be employed to enhance the separation of such proteins:   Ion Exchange Chromatography This method exploits differences in the net surface charge of proteins to achieve separation using charged chromatographic resins. By carefully selecting the type of ion exchange medium (e.g., ca......

• • What Are the Primary Factors Contributing to Chromatographic Peak Broadening?

  Chromatographic peak broadening refers to the dispersion of an analyte band along the retention time axis and significantly affects both separation efficiency and quantitative accuracy. Several key factors contribute to peak broadening:   Diffusion Processes 1. Molecular Diffusion Random motion of analyte molecules between the stationary and mobile phases causes dispersion. This includes longitudinal diffusion (along the direction of flow) and transverse diffusion (perpendicular to the flow), both of ......

• • What Is the Principle of Mass Spectrometry and How Should Beginners Conduct Mass Spectrometric Analysis?

  Mass spectrometry (MS) is an analytical technique used to identify and quantify chemical components in a sample by measuring the mass-to-charge ratio (m/z) of ionized molecules. The fundamental principle involves ionizing the molecules of interest, accelerating and separating the resulting ions based on their m/z, and detecting them to produce a mass spectrum. By analyzing the mass spectral data, researchers can determine molecular weights, structural features, and relative or absolute concentrations ......

• • How Can Data Exported from MaxQuant Be Used to Visualize Protein Mass Spectrometry Results?

  MaxQuant is a widely used software for protein mass spectrometry data analysis, enabling the processing of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data for protein identification and quantification. The data exported from MaxQuant typically include information on protein and peptide identifications as well as protein quantification. The following sections outline recommended strategies for visualizing protein mass spectrometry results using data obtained from MaxQuant:   Data Organiz......

• • What Are the Procedures for Blue Native PAGE and Two-Dimensional BN-SDS-PAGE, and How Are Anodic and Cathodic Buffers Used?

  Blue Native PAGE (BN-PAGE) 1. Experimental Materials and Buffers (1) Sample buffer (cathodic buffer): 50 mM Bis-Tris (pH 7.0), 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S, and an appropriate amount of Coomassie Blue G-250 to confer a uniform negative charge on protein complexes. (2) Loading buffer (anodic buffer): 5% Coomassie Brilliant Blue G-250, 500 mM 6-aminohexanoic acid, 100 mM Bis-Tris (pH 7.0). (3) Gel solutions and running buffer for BN-PAGE: Typically composed of 15 mM Bis-Tris (pH 7.0)......

• • What Are the Underlying Principles of Common Methods for Detecting Specific Proteins in Cells and Tissues?

  A variety of techniques are available for detecting specific proteins within cells and tissues. Below are several widely used methods, along with the fundamental principles underlying each:   1. Western Blotting This method employs specific antibodies to detect target proteins. Proteins are first extracted from cell or tissue samples and separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The separated proteins are then transferred onto a membrane, which is incubated wit......