Protein Analysis FAQ

• • Can Flow Cytometry Achieve Absolute Protein Quantification? What Methods Accurately Measure Two Membrane Proteins per Cell?

  Flow cytometry is a powerful technique for analyzing and sorting cells based on specific markers on their surface or within the cytoplasm. While it is highly effective for qualitative assessment and relative quantification—allowing comparisons of protein expression levels across different samples—it is generally not used for absolute quantification. To achieve accurate quantification of membrane proteins, particularly to determine the absolute number of molecules on the surface of a single cell, the f......

• • How Can Proteomics Be Conducted When the Complete Genome Sequence of a Species Is Available?

  When the complete genome sequence of a species is known, the following workflow outlines the fundamental steps for conducting proteomic studies:   Protein Prediction and Annotation The process begins with gene prediction from the genome sequence to identify potential open reading frames (ORFs) that encode proteins. This can be achieved using gene prediction tools such as Genscan, AUGUSTUS, or FGENESH. The predicted gene and protein sequences are then subjected to functional and structural annotation. ......

• • Why Do Phosphate-Based Buffers Hinder Protein Detection by Mass Spectrometry? Is It Due to Chelation by Phosphate Ions?

  Phosphate-based buffer systems often pose significant challenges in protein analysis by mass spectrometry (MS), primarily due to their incompatibility with several key steps and principles of the technique. The following are some of the main reasons:   Ion Suppression Phosphate buffers typically contain high concentrations of salts, which can cause ion suppression during MS analysis. Excessive salt ions compete with target peptides or proteins for ionization, thereby reducing ionization efficiency and......

• • How Can DIA Mass Spectrometry Data Be Analyzed to Obtain the Peak Trace of a Specific Peptide?

  The analysis of Data-Independent Acquisition (DIA) mass spectrometry data is inherently complex and involves multiple stages to accurately extract the peak trace of a specific peptide. The following comprehensive workflow outlines the key steps required to identify and visualize the chromatographic peak of a target peptide from DIA datasets:   1. Data Preprocessing (1) Raw data conversion: The instrument-generated raw files (e.g., .raw) are converted into standard open formats such as mzML or mzXML us......

• • What Factors Influence Protein Stability During the Purification Process?

  During protein purification, various factors can affect the structural integrity and functionality of proteins, thereby compromising their stability and overall quality. The following are commonly recognized factors that influence protein stability:   pH Variations in pH can alter the net charge of a protein, which in turn affects its solubility and conformational stability.   Temperature Elevated temperatures may cause protein denaturation and aggregation, leading to a loss of structural integrity an......

• • How Should One Address the Presence of Equivalent Bands in the Control Group After Bead Incubation in Pull-Down Assays?

  In pull-down assays, the observation of protein bands in the control group—following incubation with magnetic beads—that are comparable in intensity to those in the experimental group typically indicates non-specific binding or background interference. The following strategies may be employed to mitigate such issues:   Optimize Incubation Conditions Adjust the concentrations of both the protein sample and the magnetic beads, and experiment with shortening or extending the incubation time to minimize n......

• • Why Do Two Samples Differ in CD but Not in UV Spectra (P > 0.05)? Which Result Is More Reliable?

  When interpreting discrepancies between circular dichroism (CD) and ultraviolet (UV) spectroscopy results, it is important to consider the type of structural information each technique provides as well as their respective sensitivities. The following explains in detail the possible causes of such discrepancies and offers guidance on how to interpret the results appropriately:   Circular Dichroism (CD) vs. Ultraviolet (UV) Spectroscopy 1. Circular Dichroism (CD) (1) Far-UV CD (190–250 nm): Primarily us......

• • Why Does SUMO-Tagged Protein Fail to Undergo On-Column Cleavage on a Nickel Affinity Column?

  This issue may arise due to several contributing factors:   Insufficient Enzyme Activity The protease used for cleavage may exhibit low activity or may have lost its activity entirely. Enzyme performance can be affected by various factors such as storage conditions, temperature, and pH. It is recommended to verify the enzyme’s quality and storage conditions, or to use freshly prepared enzyme stocks.   Inadequate Enzyme Concentration The amount of enzyme added may be insufficient to ensure efficient in......

• • What Are the Methods for Deglycosylating Glycoproteins?

  Deglycosylation of glycoproteins refers to the process of removing carbohydrate moieties from glycoprotein molecules. Several established methods are commonly employed in laboratory settings to achieve this, broadly categorized into enzymatic and chemical approaches:   Enzymatic Deglycosylation 1. Endoglycosidases Enzymes such as PNGase F are widely used for cleaving most N-linked glycans from glycoproteins with high specificity and efficiency.   2. Exoglycosidases Enzymes like sialidases (also known ......

• • What Are the Detailed Experimental Procedures for Glycosylation Analysis?

  The experimental workflow for glycosylation analysis can vary significantly depending on the specific analytical goals and selected methodologies. Mass spectrometry (MS) is a powerful tool capable of providing in-depth information regarding glycosylation sites and glycan structures. Here, we present a representative protocol for the analysis of protein N-glycosylation using MS-based techniques:   Sample Preparation 1. Protein Extraction Total proteins are extracted from biological samples such as cell......