Protein Analysis FAQ

• • How to Use Asp-N or Glu-C Alone or Combined for Protein Digestion? Dosage vs. Trypsin’s 30:1 Ratio

  The following guidelines outline recommended protocols for the usage and dosage of Asp-N and Glu-C in protein digestion experiments: Using Asp-N or Glu-C Alone 1. Asp-N Dosage: A protein-to-enzyme ratio of 50:1 is recommended. Digestion Conditions: Incubate at approximately pH 8.0 and 37°C, typically overnight. 2. Glu-C Dosage: A protein-to-enzyme ratio in the range of 20:1 to 30:1 is recommended. Digestion Conditions: Optimal at pH 7.8, incubated at 37°C overnight. Sequential Digestion with Asp-N and......

• • What’s the Protocol for Detecting O-Glycosylation by WB Antibodies

  The basic steps for O-glycosylation Western Blot (WB) antibody detection are as follows: 1. Sample Preparation Collect and prepare cell or tissue samples containing the target protein. Lyse the samples using an appropriate lysis buffer, followed by centrifugation to remove insoluble debris. 2. Protein Concentration Determination Determine the protein concentration using a BCA protein assay kit or a comparable method. 3. SDS-PAGE Select an appropriate polyacrylamide gel concentration based on the molecular..

• • How Can the Presence of a Specific Receptor Protein on a Cell Be Characterized?

  Characterizing the presence of a specific receptor protein on a cell is essential for verifying and quantifying its expression on the cell surface or within intracellular compartments. Several widely adopted techniques are employed to achieve this, including:   Flow Cytometry Flow cytometry enables the quantitative analysis of receptor expression levels and the proportion of receptor-positive cells. This is achieved by labeling the target receptor with fluorescently tagged, receptor-specific antibodie......

• • How to Use Library Search Results to Identify Compounds in Mass Spectra?

  Library Search Results refer to the output generated by spectral database software when querying compound identities corresponding to peaks in a mass spectrum. To identify the chemical species represented by specific peaks using Library Search Results, the following procedure can be followed:   Acquisition of Mass Spectrometry Data First, analyze the sample using a mass spectrometer (e.g., GC-MS or LC-MS) to acquire the mass spectral data.   Processing of Mass Spectrometry Data Process the acquired da......

• • 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......