Protein Analysis FAQ

• • When to Boil the Protein in WB Experiments

  "Protein denaturation by heat, often referred to as 'boiling the sample,' involves heating a protein sample at high temperatures to induce denaturation. Typically, this process includes mixing the protein sample with a loading buffer containing SDS and a reducing agent like β-mercaptoethanol or DTT, followed by heating in a boiling water bath for 5-10 minutes. After completing the sample extraction and thorough mixing with the loading buffer, heat-induced denaturation is generally carried out prior to......

• • What Are Protein N-Linked Glycosylation and O-Linked Glycosylation

  Protein glycosylation is a crucial biochemical process in living organisms, involving the covalent attachment of one or more sugar molecules, specifically monosaccharides, to proteins. Glycosylation is typically categorized into two main types: N-linked glycosylation and O-linked glycosylation. These processes are of significant interest in cell biology, pathobiology, and biochemical research.

• • How to Determine Protein Purity Based on Electrophoresis Bands?

  Gel electrophoresis, especially SDS-PAGE, is a common technique for separating and analyzing protein mixtures. Through this technique, researchers can separate proteins based on their size and assess protein purity by examining the band pattern on the gel after electrophoresis:   Density Samples with higher purity typically show a single, thick band. If the sample contains multiple proteins, multiple bands may be observed.   Band Clarity High-purity protein bands have clear and well-defined edges. Blu......

• • 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 ......