Protein Analysis FAQ

• • Why Do Large Molecular Weight Protein Bands Appear Wavy in Western Blot

  In proteomics research, Western Blot is a common method for measuring protein expression levels. A common issue observed in Western Blot experiments is the wavy appearance of large molecular weight protein bands (also known as the "smile effect"). This can be caused by several factors: 1. Electrophoresis Conditions (1) Voltage Settings: If the electrophoresis voltage is too high, it may cause proteins to migrate too quickly, leading to wavy bands. (2) Electrophoresis Time: Both overly long and short........

• • Why Are Genes Not Displayed on the Network Graph When Analyzed with STRING

  STRING is a commonly used protein-protein interaction (PPI) database that allows users to generate PPI networks by inputting a set of protein or gene names. If the genes you entered do not appear on the network graph, there could be several reasons: 1. Gene Name Not Recognized or Mismatched The gene names you entered may be misspelled or use incorrect naming conventions, preventing STRING from recognizing them. 2. Missing PPI Data for Genes While STRING contains a vast amount of PPI data, not all genes.....

• • How to Detect Protein Expression in Cells During Experiments

  To detect a protein in cell experiments and determine whether it is expressed, various techniques can be used: 1. Western Blot A common method to detect proteins. First, separate cell proteins by gel electrophoresis, then transfer them to a membrane. Use a specific antibody to detect the target protein. If the target protein is present, the antibody will bind to the corresponding spot on the membrane, forming a band. The presence of this band confirms protein expression. 2. Immunofluorescence Using.........

• • Why Does High-Performance Liquid Chromatography Data Show Extraneous Peaks

  HPLC data showing extra peaks may stem from several causes. Here are common possibilities and their solutions: 1. Impurities in the Sample Impurities or residual solvents in the sample can lead to contaminant peaks. Purify and treat the sample before injection to minimize impurities. 2. Residual Reagents and Solvents Reagents and solvents left in parts like the column, syringe, or tubing from previous analyses may cause extra peaks. Thoroughly clean the chromatography system before each run.

• • How to Manually Integrate Peak Area on HPLC

  High-performance liquid chromatography (HPLC) is usually equipped with data processing software for real-time or offline chromatographic data analysis. In the data processing software, peak area can be integrated automatically or manually. Below are the general steps for manually integrating peak area in HPLC data processing software: 1. Open Chromatographic Data Open the chromatographic data file that needs manual integration in the data processing software. 2. Select Manual Integration Mode In the........

• • How to Calculate Protein Expression Levels in Proteomics

  In proteomics research, common methods to measure protein expression levels are based on relative or absolute quantification using mass spectrometry data. Common quantification methods include labeling and label-free approaches: 1. Labeling Quantification Proteins or peptides are labeled using isotope tagging techniques, such as SILAC, ICAT, TMT, and iTRAQ. The relative change in protein expression is calculated by comparing the signal intensity of isotope labels in different samples. Log2-fold change......

• • What Are the Forms of Protein Modifications and How Do They Affect Cell Signaling

  Types of Protein Modifications 1. Phosphorylation and Dephosphorylation Phosphorylation involves adding or removing phosphate groups to specific amino acid residues (usually serine, threonine, or tyrosine) on proteins. This is the most common form of protein modification, affecting protein structure and function, playing a key role in many signaling pathways. 2. Ubiquitination Ubiquitin proteins are linked to the lysine residues of target proteins through a system of three enzymes.

• • Protein Analysis: Can Protein WB Expression Be Verified by qPCR

  Western Blot (WB) expression of proteins and qPCR (quantitative polymerase chain reaction) actually analyze different levels of biological information. WB is mainly used to detect protein expression levels, while qPCR is used to detect mRNA expression levels of genes. Although there is some correlation between them, they measure different biomolecules. Western Blot (WB) is an immunoassay technique used for qualitative and quantitative analysis of target protein expression levels in cells or tissues.

• • Why Does the Protein Purification Process Always Fail to Bind to the Column

  During protein purification, if the protein is not effectively binding to the column, there could be several reasons: 1. Inappropriate Buffer pH Protein binding usually depends on its isoelectric point and the pH of the buffer. If the pH is not suitable, the protein may not bind correctly. 2. Salt Concentration Too High or Too Low Salt concentration affects protein solubility and binding ability. If the salt concentration is not appropriate, it may hinder protein adsorption. 3. Column Issues The column.....

• • GC-MS and LC-MS Sample Requirements: Can Cell Fermentation Broth Be Used

  GC-MS and LC-MS are commonly used analytical techniques for identifying and quantifying compounds in complex samples. However, due to differences in their working principles and application fields, their sample requirements vary. GC-MS Sample Requirements 1. Volatility Samples for GC-MS analysis must be volatile, as they need to be distilled into the gas column at high temperatures. 2. Stability Samples must be stable at the high temperatures in GC, without decomposition. 3. Purity As with LC-MS, minimize..