Protein Analysis FAQ

• • What Causes Baseline Drift During HPLC Sample Injection?

  To determine the amount of a 100-unit enzyme required for protein digestion, follow these steps:   1. Determine the Total Amount of Protein For example, if you have 1 milligram (mg) of protein.   2. Calculate the Required Enzyme Amount For Asp-N: If the recommended ratio is 50:1, the required amount of Asp-N is 1 mg / 50 = 0.02 mg.   For Glu-C: If the recommended ratio is 20:1, the required amount of Glu-C is 1 mg / 20 = 0.05 mg.   3. Convert Enzyme Amount to Units Determine how many units are present......

• • Why Is the Protein Marker Not Visible on the Membrane After Western Blot Transfer?

  This issue pertains to a common occurrence in Western blot experiments. When the protein marker (molecular weight marker) is not visible on the membrane, several factors may be responsible. The following four points are potential primary causes:   1. Selection of Protein Marker The choice of marker should be based on the molecular weight of the target protein. If the marker's molecular weight is too close to that of the target protein, or if it falls outside the target protein's range, the marker may ......

• • How to Calculate the Amount of 100 Units of Enzyme Needed for Protein Digestion?

  To determine the amount of a 100-unit enzyme required for protein digestion, follow these steps:   1. Determine the Total Amount of Protein For example, if you have 1 milligram (mg) of protein.   2. Calculate the Required Enzyme Amount For Asp-N: If the recommended ratio is 50:1, the required amount of Asp-N is 1 mg / 50 = 0.02 mg.   For Glu-C: If the recommended ratio is 20:1, the required amount of Glu-C is 1 mg / 20 = 0.05 mg.   3. Convert Enzyme Amount to Units Determine how many units are present......

• • Why Does the WB Band Appear Diffuse, Resembling Protein Separation from the Loading Control?

  When Western blot (WB) bands appear diffuse or abnormally wide, several factors may contribute to this phenomenon, giving the impression of protein separation from the loading control. The potential causes include:   1. Excessive Protein Loading Overloading the sample can cause protein diffusion within the gel, leading to broad and indistinct bands. This effect is particularly pronounced when the sample concentration exceeds the gel’s resolving capacity.   To improve band sharpness, it is essential to......

• • How to Interpret the Electrophoresis Results of SDS-PAGE for Target Protein Detection?

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for protein separation. This method relies on sodium dodecyl sulfate (SDS) to denature proteins and confer a uniform negative charge, facilitating their separation in a polyacrylamide gel. Since SDS eliminates charge-based differences, protein migration is primarily dictated by molecular weight rather than the protein’s intrinsic charge or conformation.   Interpreting SDS-PAGE Electrophoresis Results Follow......

• • How Can Protein Bands Be Analyzed in SDS-PAGE Purity Assessment?

  Band Resolution and Integrity The clarity and integrity of protein bands should be carefully examined. Diffuse or smeared bands may indicate protein aggregation, degradation, or the presence of impurities.   Number of Bands The presence of multiple bands within a single lane suggests the coexistence of multiple protein species, such as contaminants or degradation products. If the sample is expected to contain a single purified protein, only one distinct band should be observed.   Background Cleanlines......

• • An Overview of GC, GC/MS, LS, LC/MS, and ICP-MS Techniques

  This section provides an overview of various analytical techniques used for compound separation, identification, and quantification.   Gas Chromatography (GC) 1. Principle GC is primarily used for the separation and analysis of volatile compounds based on their interaction with a stationary phase under controlled temperature conditions.   2. Applications Used to determine the composition and relative abundance of volatile organic compounds in complex mixtures.   Gas Chromatography–Mass Spectrometry (G......

• • How Can Potential Modifications of a Protein Be Identified?

  Identifying potential protein modifications is typically achieved through a combination of bioinformatics analysis and experimental techniques. The commonly used approaches include:   1. Bioinformatics-Based Prediction Online databases and computational tools such as UniProt, PhosphoSitePlus, and PROSITE provide information on known post-translational modifications (PTMs) and predict potential modification sites.   Comparative sequence analysis can help identify conserved modification sites by analyzi......

• • What Techniques Are Used to Analyze Protein Structure?

  Protein structure can be studied at different hierarchical levels, including primary structure (amino acid sequence), secondary structure (local motifs such as α-helices and β-sheets), tertiary structure (the three-dimensional conformation of a single protein molecule), and quaternary structure (complexes formed by multiple interacting subunits). A variety of techniques are available for structural analysis, each with distinct advantages and limitations. The most commonly used methods include:   1. X-......

• • Why Is the FLAG Tag Not Detectable by Western Blot Despite a Strong Band Observed with the Target Antibody?

  When expressing FLAG-tagged proteins, if a strong band is detected using the target antibody but the FLAG tag is not detected using the tag-specific antibody via Western blot (WB), several factors may contribute to this issue:   1. Insufficient Specificity or Reduced Activity of the FLAG Antibody The FLAG tag antibody may have insufficient specificity or reduced activity, which could result from the antibody itself, expiration, or degradation during storage.   2. Restricted Accessibility of the FLAG T......