Protein Analysis FAQ

• • Can Ultraviolet Spectrophotometry Be Used for the Quantitative Analysis of Amino Acids and Proteins

  Ultraviolet spectrophotometry (UV-Vis spectrophotometry) is a widely employed technique for the quantitative analysis of amino acids and proteins. 1. Quantitative Analysis of Amino Acids Certain amino acids, such as tryptophan and tyrosine, possess intrinsic ultraviolet absorbance, particularly around 280 nm. Therefore, UV-Vis spectrophotometry can be used directly for their quantitative determination. In contrast, amino acids that lack UV absorbance require chemical derivatization or alternative ..........

• • Why Does Purified Protein Show 60kDa Instead of the Expected 107kDa During SDS-PAGE

  If a purified protein displays a significantly lower apparent molecular weight on SDS-PAGE than expected (e.g., expected 107 kDa but observed as 60 kDa), several potential explanations should be considered: 1. Protein Degradation The most common explanation is partial degradation of the protein during extraction or purification, often caused by endogenous protease activity. To prevent this, protease inhibitors should be added during protein extraction and purification, and all procedures should be carried..

• • Why Do Two Bands Appear in SDS-PAGE After Co-IP to Detect Protein-Protein Interaction

  Co-immunoprecipitation (Co-IP) is a widely used method for detecting protein-protein interactions. In brief, when researchers hypothesize that two proteins may interact, they can use an antibody specific to one of the proteins to immunoprecipitate it along with any proteins bound to it. The precipitated protein complex is then subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), followed by analysis through Western blotting. The appearance of two bands on the SDS-PAGE gel......

• • Is It Necessary for Proteins Used in Co-Immunoprecipitation to Have Tags Like Flag or HA

  In co-immunoprecipitation (Co-IP) experiments, it is typically necessary to use specific antibodies to recognize and isolate the target protein. These antibodies bind to the target protein and allow for its co-precipitation along with associated interacting proteins. In some cases, to improve the efficiency and specificity of Co-IP, the target protein may need to be engineered to carry specific epitope tags, such as the FLAG tag or HA tag. These epitope tags facilitate the isolation of the target protein...

• • Why Can Polyacrylamide Gel Electrophoresis Separate More Protein Bands

  Polyacrylamide gel electrophoresis (PAGE) is a widely used biochemical technique that enables the separation of proteins primarily based on their molecular weight or charge. The following factors contribute to the ability of PAGE to separate a greater number of protein bands: 1. Molecular Sieving Effect The pore size of polyacrylamide gels can be precisely controlled by adjusting the gel concentration. High concentrations create smaller pores suitable for resolving proteins with low molecular weight........

• • What Elution Buffer Should Be Used for Beads in Co-IP Samples Before Mass Spectrometry

  For Co-IP samples intended for mass spectrometry analysis, it is essential to ensure that the elution buffer does not contain components that may interfere with mass spectrometry. In general, the following types of elution buffers can be employed: 1. High-Salt Concentration Solutions A typical example is 2 M NaCl, which can reduce ionic interactions between the protein and the beads. 2. SDS-PAGE Sample Buffer This buffer can be used to directly elute proteins for subsequent separation via SDS-PAGE........

• • Why Is My Loading Control Clear in WB but the Target Protein Always Faint

  In Western blot (WB) experiments, it is sometimes observed that the loading control—typically a stably expressed protein such as GAPDH or β-actin—displays a strong and consistent signal, whereas the target protein of interest appears faint or barely detectable. Several potential factors could contribute to this discrepancy. Below, I outline common causes and suggest possible troubleshooting strategies. Antibody-Related Issues 1. Insufficient Antibody Specificity If the primary antibody cross-reacts with....

• • Can't Deduce Fragmentation from Triple Quadrupole Scan? Try Mass Frontier Software

  Mass Frontier enables the elucidation of fragment ion origins by searching databases of known molecular structures and predicting potential fragmentation pathways through its integrated library of fragmentation rules. The general workflow is as follows: 1. Import Mass Spectrometry Data Begin by importing data acquired from a triple quadrupole mass spectrometer into Mass Frontier. Ensure that the file format is supported by the software, such as mzXML, mzData, or NetCDF formats. 2. Data Processing and.......

• • What Determines Peak Width at the Baseline in HPLC

  In liquid chromatography (LC), the peak width at the baseline is governed by various factors, which mainly include: Longitudinal Diffusion During LC, the mass transfer of analyte molecules between the mobile and stationary phases results in longitudinal diffusion, a key contributor to peak broadening. This effect is particularly pronounced when using columns with low efficiency or under high-speed separation conditions. Radial Diffusion The radial (transverse) diffusion of analyte molecules within the......

• • Why Does Protein Mass Spectrometry Have Polyethylene Glycol Contamination

  Polyethylene glycol (PEG) is a widely used polymer known for its excellent solubility, biocompatibility, and non-toxicity. In biological and protein research, PEG is frequently employed in protein purification, crystallization, and as a solvent substitute. However, during protein mass spectrometry (MS) analysis, the presence of PEG can lead to contamination that compromises the accuracy and reliability of the results. The primary sources of PEG contamination are as follows: 1. Contamination from Reagents...