Protein Analysis FAQ

• • Why Do Two Proteins with Different Hydrophobicity Elute Oppositely in RPC and HIC

  To address this question, it is essential to first understand the fundamental principles of Reverse Phase Chromatography (RPC) and Hydrophobic Interaction Chromatography (HIC). Reverse Phase Chromatography (RPC) 1. Principle In Reverse Phase Chromatography, the stationary phase (i.e., column packing material) is non-polar (e.g., C18), while the mobile phase (elution buffer) is relatively polar, typically consisting of a mixture of water and an organic solvent such as acetonitrile or methanol. Under.........

• • Considerations for Protein Sample Preparation

  Protein sample preparation is a crucial procedure that warrants meticulous attention, as it has a direct impact on downstream analyses and experimental outcomes. The following considerations should be taken into account during protein sample preparation: 1. Purity It is essential to confirm that the isolated protein corresponds to the target protein and possesses a high degree of purity. Analytical techniques such as SDS-PAGE can be employed to assess protein purity. 2. Concentration Protein concentration..

• • Principles of Protein Sample Preparation Techniques

  Protein sample preparation is an essential procedure in laboratory research, aiming to obtain proteins that are pure, functionally active, and suitable for downstream experimental analyses. The following outlines several fundamental principles of protein sample preparation techniques: 1. Consistency in Sample Handling Maintaining consistency in sample handling throughout the entire experimental process is critical to minimizing human-induced variability. All samples, including both control and........

• • What Are the Methods for Detecting Protein Phosphorylation

  The following are some commonly used methods for detecting protein phosphorylation: 1. Immunoblotting (Western Blotting) Immunoblotting is a widely utilized technique for analyzing the phosphorylation status of proteins. Protein samples are first separated by electrophoresis and subsequently transferred to a membrane. Phosphorylated proteins are then identified using phosphorylation-specific antibodies. Signal detection is typically achieved via chemiluminescence or staining, allowing assessment of both....

• • What Is the Principle Behind GC-MS

  Gas Chromatography–Mass Spectrometry (GC-MS) is an analytical chemistry technique primarily used for the separation, identification, and quantification of compounds in complex samples. GC-MS integrates the functionalities of gas chromatography (GC) and mass spectrometry (MS) in a tandem configuration, offering highly selective and sensitive analysis. This technique is widely employed in environmental analysis, forensic science, food safety, drug testing, and a variety of other application areas.

• • How to Interpret the Western Blot for Protein Phosphorylation

  Western blot (WB) is a widely used technique for analyzing protein expression levels and post-translational modifications. When using phospho-specific antibodies to detect protein phosphorylation, the results can be interpreted from the following perspectives: 1. Check Background Clarity Begin by ensuring that the membrane exhibits a low background signal, allowing bands to be clearly distinguished. Excessive background may result from non-specific binding or insufficient blocking.

• • Why Is There a Step in Western Blot That Requires Determining the Protein Concentration in the Tissue

  Determining the protein concentration of tissue samples serves the following key purposes: 1. To Quantify the Relative Protein Content of Samples Measuring the protein concentration allows for accurate assessment of the relative protein content across samples. In Western blot experiments, samples from different groups—such as control and experimental—are often compared. Knowing the protein concentration ensures that equal amounts of protein are loaded into the gel, allowing for valid comparisons during.....

• • WB Shows 20kDa Increase, How to Use Dephosphorylation and Deglycosylation to Confirm Post-Translational Modifications

  In Western blot (WB) experiments, an observed increase of approximately 20 kDa in the molecular weight of a target protein compared to its predicted mass may suggest the occurrence of post-translational modifications. To verify this possibility, the following steps can be taken: Dephosphorylation Verification 1. Sample Preparation Ensure that a sufficient amount of protein sample is available for analysis. 2. Selection of an Appropriate Phosphatase Alkaline phosphatases, such as Lambda protein phosphatase..

• • Why Are There So Many Keratins in My Mass Spectrometry Results

  The frequent detection of keratins in mass spectrometry results is often attributed to sample contamination during experimental procedures. Keratins are a group of abundant and stable structural proteins commonly found in the epidermis, hair, nails, and other tissues of humans and animals. Owing to their high abundance and resilience, keratins can be readily introduced into samples throughout the experimental workflow, resulting in their pervasive presence in mass spectrometry data. The primary sources.....

• • Can WB Without SDS Work for Small Proteins (10KD)? Is Tris Used Instead for Gel Balance

  Small proteins (10 kDa) are typically analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in Western Blot (WB) experiments. SDS, as a surfactant, denatures proteins and imparts a uniform negative charge, enabling their separation based solely on molecular weight. In the absence of SDS during electrophoresis, proteins are not fully denatured. As a result, their migration behavior is influenced not only by molecular size but also by their native conformation and intrinsic charge, leading to......