Protein Analysis FAQ

• • What Are the Common Post-Translational Modifications of Proteins? Provide Four Examples

  Post-translational modifications (PTMs) are covalent modifications that occur after a protein has been synthesized from mRNA into a polypeptide chain. These modifications play a crucial role in determining protein function, activity, stability, and subcellular localization. The following are four representative types of post-translational modifications: 1. Phosphorylation Phosphorylation is a pivotal post-translational modification that involves the covalent attachment of a phosphate group (PO₄³⁻) to ......

• • A Gene Is Missing in the String: How to Proceed with Protein-Protein Interaction Analysis

  When a specific gene is absent from the analyzed DNA sequence, yet protein-protein interaction analysis remains necessary, the following strategies can be implemented: 1. Reassessing the DNA Sequence It is essential to verify the accuracy and completeness of the DNA sequence. In some cases, sequencing errors or omissions may prevent the identification of the target gene. Reanalyzing the sequence using various bioinformatics tools and databases can help detect missing or misannotated genes. 2. Identifying...

• • How to Use Chromatography to Purify Enzymes and Determine the Target Protein

  Sample preparation: The cells or tissues containing the target enzyme are lysed to obtain the crude cell extract. Ultrasonication, high-pressure homogenization, or other methods are commonly used to lyse the cells. Centrifugation is used to remove cell debris and unbroken cells after lysis. Preliminary purification: Fractionation methods (such as ammonium sulfate precipitation) are used to separate most proteins from the cell extract, resulting in crude enzyme preparation. Gel filtration chroma......

• • Are the Data Obtained from LC-MS and Proton NMR the Same

  LC-MS (Liquid Chromatography-Mass Spectrometry) and ¹H NMR (Proton Nuclear Magnetic Resonance) are two distinct analytical techniques that yield fundamentally different types of data. LC-MS (Liquid Chromatography-Mass Spectrometry) 1. Function LC-MS is used to separate various components within a sample and determine their respective molecular weights. 2. Data It generates a mass spectrum that depicts the intensity of ions at different mass-to-charge ratios (m/z). 3. Advantages LC-MS enables the ........

• • Why Is Protein Sequencing Mainly Based on Acid Hydrolysis and Supplemented by Alkaline Hydrolysis

  Protein sequencing predominantly relies on acid hydrolysis as the primary method, with alkaline hydrolysis serving as a supplementary approach for the following reasons: Higher Efficiency of Acid Hydrolysis Compared to alkaline hydrolysis, acid hydrolysis more efficiently cleaves peptide bonds, facilitating the breakdown of proteins into smaller peptides. This makes it the preferred method in protein sequencing. Under acidic conditions, peptide bonds are more susceptible to cleavage, generating peptide.....

• • What to Do If Excess Buffer Is Added During WB

  In Western Blot (WB) experiments, excessive buffer addition may adversely affect protein transfer efficiency, antibody binding, or signal detection. The appropriate corrective measures depend on the specific experimental stage at which the excess buffer was introduced. 1. Sample Preparation Stage If an excessive amount of lysis buffer or other sample preparation buffers is added: (1) Dilution: Additional sample material may be introduced to restore the intended buffer-to-protein ratio, provided this does...

• • Method for Methylation and Uronic Acid Reduction of Samples Before GC-MS Analysis

  Methylation and uronic acid reduction are essential preparatory steps before analyzing glycosidic bonds using gas chromatography-mass spectrometry (GC-MS). Methylation prevents the degradation of glycosidic bonds during subsequent analysis by replacing hydroxyl (-OH) groups in sugar molecules with methyl (-CH₃) groups, thereby enhancing molecular stability. Uronic acids, such as glucuronic acid, may exhibit instability under GC-MS conditions and are therefore typically reduced to their corresponding .......

• • How to Analyze the Glycosidic Bond Structure Using GC-MS Detection

  Mass spectral analysis is the primary approach for interpreting glycosidic bond structures when using gas chromatography-mass spectrometry (GC-MS). The following key steps should be considered for result interpretation: 1. Analysis of GC Chromatogram Peaks The peaks in the GC chromatogram represent different compounds and must be identified. Each peak corresponds to a mass spectrum, which provides information on the mass distribution of the detected compound. 2. Detailed Mass Spectral Analysis Molecular....

• • What Are the Possible Reasons for the Molecular Weight of Proteins Detected by SDS-PAGE Being Higher Than Expected

  When analyzing proteins using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), the observed molecular weight may exceed the expected value due to several factors: 1. Protein Aggregation Under certain conditions, proteins can form dimers or larger aggregates. This may result from the intrinsic biochemical properties of the protein or from specific conditions during sample preparation, such as variations in pH, temperature, and ionic strength. If aggregation occurs, the migration rate....

• • SDS-PAGE Measures Protein Molecular Weight: How Is Migration Distance Measured

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for determining protein molecular weight. Following SDS-PAGE, the molecular weight of proteins can be estimated by measuring their migration distance within the gel. The migration distance of proteins can be determined using image analysis software (e.g., ImageJ) or a ruler by measuring the distance from the sample well to the protein band. Estimating Protein Molecular Weight Using Migration Distance: .......