Protein Analysis FAQ

• • Why Can Primary Antibodies Still Recognize SDS-Denatured Proteins on Nitrocellulose in Western Blot

  In Western blotting, primary antibodies are sometimes able to recognize proteins on nitrocellulose membranes even after these proteins have been denatured by SDS. Several possible mechanisms may account for this phenomenon: 1. Structural Properties of Proteins After Membrane Transfer and Binding Upon transfer to the nitrocellulose membrane, proteins bind tightly to the surface. This interaction may induce partial refolding of the proteins, allowing some secondary or tertiary structures to recover to a......

• • Why Does the Observed Protein Run Smaller Than Predicted in Western Blot

  When a protein appears at a lower molecular weight than predicted in a Western blot (WB) experiment, several potential factors may be responsible: 1. Protein Modifications Proteins within cells may undergo various post-translational modifications such as phosphorylation, methylation, or acetylation. These modifications can alter the protein’s charge, conformation, or molecular weight, thereby influencing its electrophoretic mobility. In some cases, such modifications may increase the protein’s migration....

• • How to Analyze Protein Identification Data and Choose the Best Result from Dozens of Proteins

  Protein identification is typically performed using mass spectrometry, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS). The main types of data obtained from protein mass spectrometric analysis include: 1. Peptide Mass Spectra Peptides resulting from proteolytic digestion are analyzed by mass spectrometry to determine their mass-to-charge ratios (m/z). 2. Fragment Ion Spectra Selected peptides are further fragmented, and the m/z values of the resulting fragment ions are measured.

• • What Are the Detection Methods for Direct Interaction Between Two Proteins

  When two proteins directly interact, such interactions can significantly influence their biological functions. In biopharmaceutical research and biotechnology, identifying and characterizing these protein–protein interactions is essential for understanding molecular mechanisms and for drug development. Below are several commonly employed methods for detecting direct protein–protein interactions: 1. Co-Immunoprecipitation (Co-IP) Co-IP is a well-established technique for detecting protein–protein .........

• • Which Instruments Can Visualize Fluorescent Proteins on Native PAGE Without Staining? What Are the Common Markers

  Native PAGE (polyacrylamide gel electrophoresis) is a technique for protein separation that preserves the native structure of proteins, as it does not involve denaturation or staining. In this method, fluorescent proteins can be directly visualized using fluorescence imaging systems. Additionally, several specific proteins are commonly employed as markers for Native PAGE. Instruments Used Fluorescent proteins in Native PAGE can be directly visualized using fluorescence imaging systems. These systems........

• • What Are the Reasons for Mass Spectrometry Failing to Detect Compounds

  Mass spectrometry analysis may fail to obtain valid results due to a variety of reasons. The following are some possible reasons why mass spectrometry may fail to detect compounds: 1. Improper Sample Preparation During the process of sample preparation, problems such as sample loss, non-uniformity, or contamination may occur, leading to a too-low concentration of effective components or making them undetectable. 2. Incompatible Ion Source Different types of ion sources are suitable for different samples....

• • How is Mass Spectrometry Used to Study Proteins

  Mass spectrometry (MS) is a powerful analytical technique widely employed in protein research to investigate mass, structure, post-translational modifications, and molecular interactions. The following are the primary approaches for studying proteins using mass spectrometry: 1. Protein Mass Determination MS enables precise measurement of protein molecular weight, which facilitates assessment of protein purity and molecular consistency. This analysis is typically performed using techniques such as matrix-...

• • How to Determine Protein Phosphorylation Sites

  Determining protein phosphorylation sites requires a combination of biological experiments and bioinformatics analyses. Together, these approaches enable the accurate identification and validation of phosphorylation sites on specific proteins, which is critical for understanding protein functions and for elucidating the regulation of protein–protein interactions. Experimental Methods 1. Mass Spectrometry Analysis Mass spectrometry is one of the most widely used approaches for the identification of protein..

• • What Are Protein Post-Translational Modifications and Their Biological Functions

  Post-translational modifications (PTMs) refer to chemical and functional alterations that occur in proteins following ribosomal synthesis. These modifications are mediated by the addition of chemical groups, cleavage of peptide bonds, conformational changes, or interactions with other biomolecules such as proteins, nucleic acids, or lipids. The major types of PTMs, along with their mechanisms of action and biological functions, are outlined below:

• • How to Use Asp-N or Glu-C Alone or Combined for Protein Digestion? Dosage vs. Trypsin’s 30:1 Ratio

  The following guidelines outline recommended protocols for the usage and dosage of Asp-N and Glu-C in protein digestion experiments: Using Asp-N or Glu-C Alone 1. Asp-N Dosage: A protein-to-enzyme ratio of 50:1 is recommended. Digestion Conditions: Incubate at approximately pH 8.0 and 37°C, typically overnight. 2. Glu-C Dosage: A protein-to-enzyme ratio in the range of 20:1 to 30:1 is recommended. Digestion Conditions: Optimal at pH 7.8, incubated at 37°C overnight. Sequential Digestion with Asp-N and......