Protein Analysis FAQ

• • What Explains the Absence of Overexpressed Proteins in Mass Spectrometry? Are Sample, Instrument, or Conditions to Blame?

  When overexpressed proteins are not detected in mass spectrometry (MS) results, multiple technical or experimental factors may be responsible. Potential causes include:   Insufficient Protein Expression The target protein may be expressed at levels too low for MS detection. Optimizing expression conditions—such as induction duration, temperature, and inducer concentration—may help enhance protein yield.   Sample Preparation Artifacts Protein degradation, loss, or denaturation during sample handling ca......

• • Why Can't Proteins Be Detected in Negative Mode Mass Spectrometry When pH Is Above Their Isoelectric Point?

  When the pH exceeds the isoelectric point (pI) of a protein, the protein tends to acquire a negative charge. This occurs because positively charged amino acid residues such as lysine, arginine, and histidine lose their protons, while the negatively charged residues like aspartic acid and glutamic acid retain their charge, resulting in an overall negative charge on the protein. Despite this, negative mode is seldom employed in protein mass spectrometry for several reasons:   Electrospray Ionization (ES......

• • What Are the Underlying Causes of Weak or Absent Protein Signals in Western Blotting?

  When weak or absent protein signals are observed in Western blotting (WB), several potential causes should be considered:   Issues Related to Sample Preparation 1. Low Protein Concentration Inadequate protein levels in the sample may fail to generate a detectable signal. This can be addressed by increasing the total protein yield, for instance, by harvesting more cells or using a larger amount of tissue to enhance protein concentration.   2. Improper Handling During Preparation Protein degradation or ......

• • What Are the Steps for Determining the Primary Sequence of a Protein and the Rationale Behind Each?

  Determining the primary sequence of a protein is typically accomplished using mass spectrometry-based proteomic approaches, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedure involves several key steps, each with a specific rationale:   1. Protein Purification The target protein must first be purified to eliminate interference from contaminating proteins during mass spectrometry. Common purification techniques include ion exchange chromatography, affinity chromatogra......

• • What Are the Limitations of Using SDS Discontinuous System Electrophoresis for Determining the Relative Molecular Mass of Protei

  SDS discontinuous system electrophoresis is a widely used method for determining the relative molecular mass of proteins. This technique employs sodium dodecyl sulfate (SDS) to bind uniformly to proteins, conferring a consistent negative charge and enabling size-dependent separation in an electric field.   However, this method presents several limitations in accurately assessing protein molecular masses:   Limited Accuracy for Low Molecular Weight Proteins SDS discontinuous system electrophoresis is p......

• • 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 ......