Protein Analysis FAQ

• • Possible Causes for Target Bands Merging into One in WB, While Internal Control is Normal?

  If your Western Blot (WB) target bands merge into a single band, but the internal control is normal, the possible causes could include:   Sample Overloading If too much of the target protein sample is loaded, the bands may merge into one. Make sure to load an appropriate amount of sample to avoid overloading.   High Antibody Concentration or Affinity If the primary or secondary antibody has too high an affinity or concentration, it may lead to over-staining and cause the bands to merge.   Excessive Mi......

• • What is the Operational Principle for Calculating Protein Sample Concentration in Western Blot (WB)?

  When using Western Blot (WB) technology to detect protein expression levels, determining the protein sample concentration is essential. This is because, in WB experiments, it is important to load the same amount of protein sample onto the gel to ensure the comparability and accuracy of results. The operational principle for calculating the protein sample concentration in WB is as follows:   Theoretical Basis The principle behind calculating protein sample concentration in WB relies on colorimetric ass......

• • What Are the Optimal Transfer Conditions for a 160 kDa Protein in WB?

  When performing Western blot (WB) analysis for a 160 kDa target protein, several key transfer conditions must be optimized to ensure efficient protein transfer onto the membrane.   1. Choice of Membrane For high molecular weight proteins such as 160 kDa, PVDF (polyvinylidene fluoride) membranes are preferred due to their superior protein-binding affinity compared to nitrocellulose membranes.   2. Transfer Buffer Composition The transfer buffer should contain methanol at a concentration of 10–20%. Meth......

• • How to Address Inconsistencies in Trends in Western Blot (WB) Experiments?

  In Western Blot (WB) experiments, inconsistencies in trends often indicate significant variability between repeated trials. To resolve this issue, the primary strategy is to thoroughly review each step of the experiment to ensure accuracy and reproducibility. Recommended measures include:   1. Sample Preparation Ensure that the protein concentrations are uniform across all samples. Use protease inhibitors to prevent protein degradation. Standardize the handling of samples during the experiment to avoi......

• • How Can MS/MS Fragmentation Information Be Utilized?

  MS/MS (Tandem Mass Spectrometry) is a powerful analytical technique that enables structural elucidation of compounds by generating secondary fragment ions through sequential fragmentation of precursor ions. By analyzing the mass-to-charge ratio (m/z) and intensity of these fragment ions, critical insights into molecular structures can be obtained. The primary applications of MS/MS fragmentation data include:   1. Structural Elucidation and Characterization By examining the m/z values and relative inte......

• • What Is the Experimental Procedure for Isoelectric Focusing?

  Isoelectric focusing (IEF) is a high-resolution electrophoretic technique that separates proteins based on their isoelectric points (pI) within a pH gradient by applying an electric field. IEF utilizes the principle that biomolecules have a net charge of zero at their pI, causing them to focus at specific locations within the pH gradient. The general protocol for IEF is as follows:   1. Sample Preparation Protein samples may require purification or processing based on experimental needs. The prepared ......

• • How Is the N-Terminal Amino Acid Position Determined? Can It Be Defined Arbitrarily?

  To accurately determine the specific amino acid sequence position of a protein's N-terminus (amino terminus), the following steps should be considered:   Identifying the N-Terminal Sequence Position The N-terminal sequence refers to the initial region of a protein or polypeptide chain, typically starting with the first amino acid residue in the sequence.   Defining the N-Terminus in Functional Domains or Signal Peptides In certain contexts, such as studies on functional domains or signal peptides, the......

• • Why Is MRM the Preferred Method for Accurate Quantification in Mass Spectrometry?

  To accurately understand why MRM is the most effective method for quantification, it is essential to define the relationship among SIM (Selected Ion Monitoring), SRM (Selected Reaction Monitoring), and MRM (Multiple Reaction Monitoring). SIM and SRM can be regarded as specific applications within the broader MRM framework. SIM is primarily employed in single quadrupole mass spectrometers, whereas SRM and MRM are mainly utilized in triple quadrupole mass spectrometers. In many contexts, SRM and MRM are......

• • Why Do Western Blot Bands Appear Inconsistent for Proteins Extracted from the Same Batch?

  When conducting a Western blot experiment, inconsistent band patterns from the same batch of extracted proteins may arise due to the following factors:   Variations in Electrophoresis Conditions Fluctuations in voltage or current can impact protein migration and separation.   Gel-Related Issues Defects in the SDS-PAGE gel, such as uneven polymerization or prolonged storage, may affect protein resolution.   Transfer Process Irregularities Non-uniform current distribution or improper transfer time and v......

• • What Are the Effects of Polarity Reversal During Protein Electrophoresis?

  In protein electrophoresis, reversing the polarity of the power supply leads to the reverse migration of proteins. Under normal conditions, proteins migrate from the cathode (negative electrode) to the anode (positive electrode) due to the applied electric field. However, if the polarity is reversed, the direction of the electric field is also inverted, causing proteins to migrate in the opposite direction. As a result, proteins may move out of the gel and diffuse into the buffer, preventing proper se......