Protein Analysis FAQ

• • How Are the B Ions and Y Ions Defined in Mass Spectrometry

  B ions and y ions are two common types of fragment ions observed in peptide analysis using tandem mass spectrometry. These ions are generated during the fragmentation process, typically through collision-induced dissociation (CID) or other fragmentation methods. B ions are generated from the amino-terminal (N-terminal) of the peptide chain. When a peptide bond breaks, and the charged fragment includes the N-terminal, the resulting ion is referred to as a b ion.

• • Method Using a DSS Crosslinker for IP and Polymeric Protein WB

  DSS (Disuccinimidyl Suberate) crosslinker is commonly used to stabilize protein-protein interactions, thereby enhancing the efficiency of immunoprecipitation (IP) and subsequent Western Blot (WB) analysis. Below is a protocol for performing IP and crosslinked protein WB analysis using DSS: Crosslinking: 1. Sample Preparation: Extract proteins from cells or tissues and adjust protein concentration. 2. DSS Addition: Add DSS crosslinker to the protein sample. DSS is a widely used reversible crosslinker that...

• • What Is the Principle of Capillary Electrophoresis Purity Analysis (CE-SDS)? What Are the Functions of the Reagents Used

  Capillary Electrophoresis (CE) is a separation technique that leverages electrophoretic principles to differentiate molecules based on their migration rates in an electric field, facilitated by applying high voltage within a narrow capillary. In CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), the SDS-PAGE technique is integrated into the capillary electrophoresis system to enable protein sample separation and purity analysis.

• • How to Calculate Protein Loading Amount for SDS-PAGE

  The goal of determining the sample loading volume is to ensure that the protein electrophoresis bands are clearly visible after SDS-PAGE, but not so concentrated that the bands become blurry. First, it is essential to know the protein concentration in your sample. This is typically achieved through protein concentration determination methods such as Bradford, BCA, or Lowry assays. These methods yield a quantifiable measurement of protein concentration, typically expressed in micrograms per microliter.......

• • Optimal Sample Loading Volume for WB Electrophoresis: Determination Factors and Consequences of Overloading or Underloading

  Western Blot (WB) electrophoresis sample loading volume is determined by several factors, including gel size, well capacity, sample concentration, and the abundance of the target protein. Typically, the loading volume ranges from 10 to 30 µL. If the loading volume is too high, the following issues may occur: 1. Sample Overflow Excessive sample may spill into adjacent wells, causing cross-contamination and misinterpretation of results. 2. Blurry Bands Overloading can lead to smeared or indistinct bands......

• • The Differences Between Channel Proteins, Carrier Proteins, and Receptor Proteins

  Channel proteins, carrier proteins, and receptor proteins are all membrane proteins that play critical roles in biological processes such as substance transport and signal transduction. However, their functions and working mechanisms differ. 1. Channel Proteins Channel proteins are proteins that form channels in the cell membrane, allowing specific molecules and ions to pass through. These channels can be either open or closed, depending on the cell’s needs. For example, potassium ion channels selectively..

• • Internal Standard Method and Calibration Curve Analysis in Gas Chromatography

  Gas Chromatography (GC) is a widely used analytical chemistry technique primarily for the qualitative and quantitative analysis of components in mixtures. In GC, the internal standard method and calibration curve method are two commonly used quantification approaches. Internal Standard Method The internal standard (IS) method is a relative quantification technique that corrects the concentration of the target analyte by adding a known concentration of an internal standard. The internal standard should......

• • If There Is Salt in the Sample, What Should Be Done on the Mass Spectrometry

  Salt in the sample can interfere with mass spectrometry analysis, compromising the accuracy of the results. If the sample contains salt, the following methods can be employed to treat it: Desalting: Desalting columns (e.g., PD-10 or Zeba Spin) or desalting centrifuge tubes can be used to remove salt from the sample. These methods rely on gel filtration to separate large biomolecules (e.g., proteins) from small salt ions, thereby effectively removing the salt.

• • Determining Relative Molecular Mass via Mass Spectrometry: The Principle of Maximum Mass-to-Charge Ratio

  When employing mass spectrometry (MS) to ascertain the relative molecular mass, the mass-to-charge ratios (m/z) of molecular ions and various fragment ions are typically observed. A molecular ion is an ion formed when a molecule loses or gains an electron without the loss or separation of other atoms or groups.In a mass spectrum, the molecular ion's m/z value in positive ion mode generally corresponds to the analyte's relative molecular mass. This is due to several factors:

• • What Concentration Should TCEP and IAA Be Used for Protein Reduction and Alkylation

  TCEP: Commonly used for reducing protein disulfide bonds. Typical concentrations range from 5 mM to 10 mM. It is important to note that the final concentration of TCEP may be adjusted based on the specific needs of the experiment and the characteristics of the protein. IAA: Used to alkylate reduced free thiols to prevent the reformation of disulfide bonds. The typical concentration of IAA ranges from 15 mM to 50 mM. Similarly, the specific concentration may need to be adjusted according to the specif......