Protein Analysis FAQ

• • What to Do If X Appears in Protein Sequence Analysis?

  In protein sequence analysis, an X usually indicates an undetermined amino acid residue at that position. X is a missing marker in the protein sequence, potentially caused by technical issues in experiments or sequencing. In some cases, X may also represent an unknown or abnormal amino acid.   When X appears in a protein sequence, consider the following points:   Determine the Missing Position First, identify the exact position of X. This can be checked through sequencing data or other experimental te......

• • How to Quantify Protein Expression in Cells Using Western Blot?

  Western blot (protein blotting) is typically used for qualitative analysis of protein presence and studying expression changes under different conditions. The general steps are as follows:   Sample Preparation Use appropriate lysis buffer to lyse the cells, and measure the total protein concentration using a protein assay method (such as BCA or Bradford).   SDS-PAGE Choose the appropriate gel based on the expected protein size and load the same amount of total protein into each lane.   Transfer Transf......

• • What Are the Basic Types, Functional Localization, and Biological Significance of Protein Glycosylation?

  Protein glycosylation (glycosylation) is a widespread biochemical process that involves the addition of sugar chains to proteins. The structure and composition of the sugar chains vary greatly, making glycosylated proteins have diverse functional properties. The following are some major types of protein glycosylation and their biological functions:   N-linked Glycosylation This is the most common type of glycosylation, involving the addition of sugar chains to the asparagine residues of proteins.   1.......

• • How to Analyze SDS-PAGE Bands for Purified GST-tagged Protein Samples?

  SDS-PAGE electrophoresis is a common protein analysis method that separates proteins based on their molecular weight. For purified GST-tagged protein samples, one or more protein bands should be visible after SDS-PAGE. The following are general steps for analyzing SDS-PAGE results:   Determine Protein Band Position First, determine the position of the GST-tagged protein on the gel. This is usually done by comparing the migration distance of the sample to a molecular weight standard. A band correspondi......

• • Why Do Proteins Get Stuck and Fail to Move Down in Western Blot?

  When performing Western blot, if proteins get stuck and fail to migrate, it could be due to several reasons:   1. Electrophoresis Conditions (1) Low Voltage: If the voltage is too low, the protein migration speed will be slow, making it seem like the proteins are stuck.   (2) Insufficient Running Time: If the electrophoresis time is too short, proteins may not have enough time to travel through the gel.   2. Gel Issues (1) Inappropriate Pore Size: If the gel’s pore size is too small, large molecular w......

• • What Software is Used for Circular Dichroism Data Analysis?

  Circular Dichroism (CD) is a spectroscopic technique used to study the structures of biological macromolecules like proteins and nucleic acids. Several software tools are available for analyzing CD data, including the following common ones:   CDPro CDPro is software for analyzing protein secondary structure. It includes three main programs: CONTIN, SELCON3, and CDSSTR, which predict protein secondary structure content based on CD data by using reference datasets.   DichroWeb DichroWeb is an online CD ......

• • What Is the Mechanism of SDS Binding to Proteins?

  The mechanism of SDS binding to proteins involves both hydrophobic and electrostatic interactions. SDS is a cationic surfactant with a long hydrophobic hydrocarbon tail and a negatively charged sulfate head. Due to its amphipathic nature, SDS can interact with many biomolecules, including proteins.   The hydrophobic tail of SDS interacts with the hydrophobic amino acid residues of the protein, unfolding its three-dimensional structure into a linear or near-linear form. This is crucial in SDS-PAGE, as ......

• • How to Prepare Samples, Concentration, and Buffer for Circular Dichroism Analysis of Protein Secondary Structure?

  Circular Dichroism (CD) is a commonly used spectroscopic technique for analyzing protein secondary structure. The preparation of samples and measurement conditions significantly impact the quality and accuracy of the CD spectra. When preparing samples, pay attention to the following:   Sample Preparation 1. Protein needs to be purified, avoiding impurities and other interfering substances.   2. If the protein needs to be refolded, ensure that the correctly folded protein is obtained.   3. Avoid using ......

• • What Are the Reasons for No Peaks in Liquid Chromatography Samples?

  Liquid Chromatography (LC) is a common separation and analysis method used to detect and quantify components in complex samples. If no peaks appear during the LC process, several reasons may be responsible:   Instrument Malfunction Certain parts of the chromatograph, such as the pump, detector, injector, or column, may be faulty. Check that the instrument is properly turned on, running, and that all connections in the LC system are intact.   Column Efficiency Prolonged use or improper handling of the ......

• • What Are the Methods for Protein Covalent Modifications?

  Protein covalent modifications refer to the non-translational addition of specific chemical groups to a protein’s amino acid residues via chemical or enzymatic reactions, thereby altering its structure and function. This is a crucial process in cellular regulation and signal transduction. Below are several methods for protein covalent modifications:   Phosphorylation A kinase-catalyzed process that adds a phosphate group to the serine, threonine, or tyrosine residues of proteins. This important regula......