Protein Analysis FAQ

• • Is There a Protocol for Blue Native PAGE and How Should the Gel Be Prepared?

  The following describes a basic procedure for preparing BN-PAGE gels. The specific parameters (e.g., concentration, pH) should be optimized according to the characteristics of the sample and the experimental objectives. Gel Solution Formulation 1. Separating Gel (12%) 30% acrylamide solution: 12 mL 1.5 M Tris-HCl, pH 8.8: 12.5 mL Deionized water: 9.4 mL 10% APS: 500 µL TEMED: 50 µL 2. Stacking Gel (4%) 30% acrylamide solution: 1.33 mL 1.0 M Tris-HCl, pH 6.8: 2.5 mL Deionized water: 6.07 mL 10% AP......

• • Prior to Mass Spectrometry Submission, Should the Protein Gel Be Stored in Deionized Water? Will the Protein Diffuse or Degrade?

  The protein gel can be stored in deionized water (ddH₂O) before submission for mass spectrometry; however, long-term storage in deionized water is not advised, as this may lead to protein dispersion into the surrounding solution or degradation. To preserve protein stability, it is recommended to select an appropriate storage method based on the specific requirements of subsequent analyses, such as short-term storage under low-temperature conditions or in a suitable buffer solution. Ideally, mass spect......

• • Which Samples Require Low Temperature, Dry Ice, or Ambient Conditions During Transportation?

  The temperature requirements for sample transportation primarily depend on the physicochemical or biological properties of the samples. The following outlines common sample categories and their corresponding temperature specifications:   1. Biological samples (e.g., blood, cells, tissues) These specimens generally require cryogenic transportation to preserve cellular viability and prevent biochemical degradation. Dry ice or liquid nitrogen is typically employed to maintain the necessary ultra-low temp......

• • What Causes Two Closely Spaced Protein Bands Before and After Enzymatic Digestion, and How Can This Be Resolved?

  Before proteolytic digestion, the protein is observed as two closely spaced bands. This pattern persists after enzymatic cleavage and subsequent AKTA purification. Several factors may account for this observation: 1. Protein Isoforms The presence of distinct isoforms or post-translationally modified variants of the protein, such as phosphorylation or glycosylation, may give rise to multiple bands of similar electrophoretic mobility. 2. Partial Degradation Partial proteolysis during protein extractio......

• • Can the Protein Precipitate Be Removed by Vacuum Filtration in the Sevag Deproteinization Method?

  The Sevag method is commonly employed to remove proteins from protein-containing liquid samples. This approach utilizes a mixed solvent of chloroform and isopropanol to denature and separate proteins, while nucleic acids such as DNA or RNA remain in the aqueous phase. During the process, proteins in the sample are denatured and aggregated by the organic solvents, forming a precipitate. This precipitate can subsequently be removed from the sample by vacuum filtration or centrifugation, thereby effectiv......

• • Which Amino Acid Serves as the Attachment Site for the Oligosaccharide Chain in N-Linked Glycosylation?

  In N-glycosylation, the oligosaccharide chain is typically covalently linked to the amide nitrogen of the side chain of an asparagine residue located within a specific amino acid sequence motif, NXS/T (where X represents any amino acid except proline), which is recognized by oligosaccharyltransferase. This glycosylation event constitutes an integral step in intracellular biosynthesis and plays a pivotal role in protein stability, correct folding, and biological function.

• • How Can Software Be Used to Quantify the Expression Levels of Ubiquitinated Bands, and Which Tools Are Recommended?

  The quantification of protein band expression following ubiquitination is typically performed using Western Blot imaging combined with densitometric analysis. Below are several widely used software tools and their respective functionalities: 1. ImageJ/Fiji An open-source platform well-suited for novice users. It enables precise measurement of band intensity and quantitative assessment of protein expression. Using this software, specific band regions can be selected, background correction applied, and......

• • At Which Stage Should Protein Concentration Be Determined After Extraction or After Reduction and Alkylation?

  Protein concentration is typically determined immediately after protein extraction, prior to any subsequent procedures such as reduction and alkylation. This practice ensures accurate determination of the initial protein concentration for use in downstream experimental steps, thereby facilitating precise calculation and adjustment of the required reagent volumes. Following reduction and alkylation procedures, protein concentration is generally not reassessed, unless specifically required by the experi......

• • How to Utilize the UniProt Protein Database to Retrieve All Glycopeptide M/Z Data of Hrp?

  To retrieve the complete glycopeptide m/z (mass-to-charge ratio) data for a specific protein (e.g., hrp) from the UniProt protein database, proceed as follows: 1. Access UniProt and Perform a Protein Search Visit the official UniProt website and enter hrp or the full name of the protein into the search bar.   2. Select the Corresponding Protein Entry From the search results, select the protein entry corresponding to your target protein and relevant to your research objectives.   3. Locate Glycosylatio......

• • How to Perform Proteomics Sample Pretreatment?

  Proteomics sample pretreatment is a crucial procedure that has a direct impact on the quality and accuracy of protein analysis. The detailed steps for proteomics sample pretreatment are as follows:   1. Protein Concentration Determination Quantify the extracted proteins using appropriate methods, such as the bicinchoninic acid (BCA) assay, Bradford assay, or other suitable colorimetric assays. This step ensures accurate preparation for subsequent experiments and quantitative analyses by maintaining co......