Protein Analysis FAQ

• • How Should a 266 kDa Protein Be Electrophoresed? What Gel Concentrations and Transfer Voltage or Current Should Be Used?

  For the electrophoresis of a 266 kDa protein, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is commonly employed. The following outlines the recommended procedure and parameter settings:   Sample Preparation The protein sample is mixed with loading buffer containing SDS and a reducing agent such as β-mercaptoethanol. The mixture is then heated in a water bath at 100°C for 5 minutes to ensure complete denaturation and dissociation of protein complexes.   Gel Preparation Polyacryl......

• • Why Does an Excessive Peak Appear Within 5 Min in Protein LC with 0.1% TFA and TFA/Acetonitrile?

  In liquid chromatography (LC) analysis for proteomics, the occurrence of a peak significantly higher than the target peak within 5 minutes may be due to several factors:   System Peaks or Baseline Drift At the initial stage of analysis, system peaks may arise due to the equilibration of the solvent components in the mobile phase with the column. This is particularly common when using mobile phases containing organic solvents (such as acetonitrile) and 0.1% trifluoroacetic acid (TFA).   Impurities in t......

• • How to Select Differential Genes for KEGG Analysis with Metabolomics and Transcriptomics Data?

  When conducting KEGG pathway annotation and enrichment analysis with metabolomics and transcriptomics data to identify differential genes, follow these steps:   Data Preprocessing 1. Transcriptomics Data (1) Quality Control: Use tools like FastQC to check raw sequence quality, then trim low-quality reads with Trimmomatic or fastp. (2) Sequence Alignment: Align clean reads to the reference genome using HISAT2, STAR, or Bowtie2. (3) Expression Quantification: Use featureCounts or HTSeq to count reads pe......

• • How to Prepare the WB Sample Loading Buffer?

  In Western Blot (WB) experiments, the sample loading buffer (Loading Buffer or Sample Buffer) typically contains the following key components:   1. Tris-HCl Buffer 0.5 M, pH ~6.8   2. SDS (Sodium Dodecyl Sulfate) 10%   3. Glycerol 20-30%   4. Bromophenol Blue 0.1-0.2% (tracking dye)   5. β-Mercaptoethanol or DTT (Dithiothreitol) 5-10%   Preparation and Usage Mix the above components in the desired proportions, adjusting the final volume as needed. Typically, the loading buffer is mixed with the sample......

• • How to Arrange Sample Order in a WB Experiment?

  When arranging sample order in a Western Blot (WB) experiment, consider the following:   1. Control Samples Place positive controls (expressing the target protein) and negative controls (not expressing the target protein) at the beginning and end for comparison and validation.   2.Replicates Arrange replicate samples side by side to facilitate comparison and analysis.   3.Loading Control Ensure each sample group includes an internal control protein (e.g., GAPDH or β-actin) for normalization.   Proper ......

• • What Are the Enzymes in Protein Glycosylation Pathways?

  Protein glycosylation includes two main types: N-linked glycosylation and O-linked glycosylation, each involving different enzymes.   N-Linked Glycosylation 1. Glycosyltransferases Transfer sugar units from a donor to specific amino acid residues on proteins.   2. Deglycosylating Enzymes Such as PNGase F, which removes glycans from proteins.   3. Peptidoglycan Transferases Assist in adding sugars to proteins in the rough endoplasmic reticulum.   O-Linked Glycosylation 1. Glycan Synthases Facilitate O-......

• • How to Analyze Node Count, Degree, and Betweenness in PPI Networks-Cytoscape or Other Software?

  Protein-Protein Interaction (PPI) network analysis can be performed using various software, including but not limited to Cytoscape.   Analyzing PPI Networks with Cytoscape 1. Import PPI Data After installing and launching Cytoscape, import PPI data via the “File” menu. Supported formats include SIF or XGMML.   2. Node Count Analysis The total number of nodes can be directly observed in the network visualization.   3. Node Degree Analysis Node degree represents the number of edges connected to a node. ......

• • What Are the Types, Principles, and Methods of Protein Electrophoresis?

  Protein electrophoresis is a technique that separates proteins under an electric field based on their size, charge, and shape. Below are common types of protein electrophoresis, their principles, and experimental methods:   Continuous SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) 1. Principle SDS, an anionic surfactant, binds to proteins, linearizing them and giving a uniform negative charge. Migration depends on size, not charge.   2. Method (1) Sample Preparation: Mix protein ......

• • How to Analyze Impurity Protein Data in BCA Assay?

  The BCA protein assay is a common method for measuring protein concentration, based on two chemical reactions: protein binds to copper ions, forming a complex that reduces bicinchoninic acid (BCA), producing a purple product with absorbance proportional to protein concentration.   1. Data Analysis Compare measured values with the standard curve to determine the protein concentration in unknown samples. Higher absorbance indicates higher protein concentration.   2. Units BCA assay results are typically......

• • How to Solve Undetectable Target Protein in Eukaryotic Overexpression WB?

  When overexpressing a target protein in a eukaryotic system but failing to detect it via Western Blot (WB), several factors may be responsible. Here are some diagnostic steps and strategies to address this issue:   Issues with Detection Method 1. Ensure correct WB procedures, including sample preparation, electrophoresis, transfer, and antibody probing.   2. Check protein sample concentration; low levels may prevent detection.   3. Verify electrophoresis and transfer efficiency to confirm protein tran......