Protein Analysis FAQ

• • Does Gel Filtration Always Elute Large Molecules First? Can Small Molecules Co-Elute? Is This Method Reliable?

  Gel filtration chromatography is a widely used protein separation and purification technique based on differences in molecular size and shape within a gel column. In this method, large molecules typically elute first, but in some cases, a small number of small molecules may co-elute with them.   Principle of Gel Filtration Chromatography Gel filtration separates molecules based on their size and shape as they pass through a gel column. The gel is usually composed of materials like polyacrylamide or ag......

• • What Protein Properties Are Characterized by the Isoelectric Point?

  The isoelectric point (pI) is a crucial parameter for characterizing proteins, reflecting the following properties:   Charge State Proteins contain ionizable functional groups, such as carboxyl and amino groups, which affect the overall charge. The pI is the pH at which the protein carries no net charge, as the number of positively and negatively charged groups are equal.   Solubility Near the isoelectric point, protein solubility is at its lowest due to minimal electrostatic repulsion, which may lead......

• • Which Method is More Accurate for Protein Structure Determination: X-ray Diffraction or NMR?

  X-ray diffraction and nuclear magnetic resonance (NMR) are two widely used techniques for protein structure determination. Both methods offer high accuracy but are suited for different applications.   X-ray Diffraction (X-ray Crystallography) X-ray crystallography is one of the most commonly used techniques for determining protein structures. It involves exposing protein crystals to X-ray beams and analyzing the resulting diffraction patterns to infer atomic positions and resolve the protein’s three-d......

• • What Is Biological Mass Spectrometry?

  Biological mass spectrometry (bio-MS) refers to mass spectrometry techniques applied in biological research, covering the identification, quantification, and structural analysis of biomolecules such as proteins, peptides, nucleic acids, and metabolites. Bio-MS has extensive applications in biological studies, including:   Proteomics Mass spectrometry enables proteome analysis, identifying and quantifying proteins in cells or tissues. Common methods include liquid chromatography-mass spectrometry (LC-M......

• • What Causes Incomplete Protein Transfer in Western Blotting?

  Protein transfer in Western blotting is the process of moving proteins from a polyacrylamide gel (SDS-PAGE) onto a membrane, typically polyvinylidene fluoride (PVDF) or nitrocellulose (NC). Inefficient transfer may lead to weak or undetectable protein signals. Several factors can contribute to incomplete protein transfer:   Suboptimal Transfer Time or Voltage Settings The optimal transfer conditions depend on protein size and gel thickness. High molecular weight proteins may require longer transfer ti......

• • Can the Same Loading Control Be Used for Two Proteins in Separate Western Blots?

  In Western blotting, loading controls (e.g., GAPDH, β-actin) are commonly used for protein normalization. If two target proteins are presented in separate blots, the same loading control can be used for normalization, provided that the following conditions are met:   Same Sample Origin Both the target proteins and the loading control must be derived from the same cell lysate or tissue extract.   Consistent Sample Processing The two target proteins must undergo identical sample processing, including bo......

• • How to Conduct WB Quantification Using ImageJ?

  The following steps outline the basic procedure for performing Western Blot (WB) quantification using ImageJ:   Importing the Image Open ImageJ and navigate to ‘File’ > ‘Open’ to load the WB image.   Adjusting Image Brightness and Contrast To ensure optimal analysis, adjust the image's brightness and contrast by selecting ‘Image’ > ‘Adjust’ > ‘Brightness/Contrast’.   Defining the Region of Interest (ROI) Use the rectangular selection tool to outline the band region you wish to quantify.   Performing A......

• • Which Three Amino Acids Are Involved in Protein Glycosylation Sites?

  Protein glycosylation can be categorized into two main types: N-glycosylation and O-glycosylation.   N-Glycosylation N-glycosylation occurs at the amide side chain of asparagine (Asn, N). N-glycosylation sites typically follow a specific sequence motif: Asn-X-Ser/Thr, where X can be any amino acid except proline due to structural constraints.   O-Glycosylation O-glycosylation typically occurs on serine (Ser, S) or threonine (Thr, T). Unlike N-glycosylation, O-glycosylation does not exhibit strict sequ......

• • Can SDS-PAGE Determine Hemoglobin's Molecular Weight?

  Yes, SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) can be used to determine hemoglobin's molecular weight. This method separates proteins based on their molecular weight, allowing for accurate determination.   Procedure 1. Sample Preparation Mix hemoglobin with SDS and a reducing agent (e.g., β-mercaptoethanol) to denature the protein and impart a uniform negative charge.   2. Gel Electrophoresis Load the prepared sample onto a polyacrylamide gel. Apply an electric current; prot......

• • Should Protein Quantification Be Done for ELISA Samples with Varying Concentrations Using BCA or Coomassie Blue?

  In ELISA experiments involving tissue samples, consistent protein concentration across samples is crucial for reliable results. Therefore, performing protein quantification on each sample is necessary to ensure uniform or known protein concentrations. Common quantification methods include BCA (Bicinchoninic Acid) and Coomassie Brilliant Blue assays.   BCA Protein Quantification 1. Based on the reaction between proteins, bicinchoninic acid, and copper ions under high temperature. 2. Less sensitive to m......