Protein Analysis FAQ
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• How to Interpret Proteomics Mass Spectrometry Data
Proteomics mass spectrometry data are obtained using a mass spectrometer to analyze protein composition and structure. Observing and interpreting mass spectra requires understanding the basic components and features of the spectra. Basic Features of Mass Spectra 1. X-axis Represents m/z (mass-to-charge ratio). 2. Y-axis Represents intensity (usually relative intensity, not directly comparable between different spectra). Common Types of Mass Spectra 1. MS1 (Primary Mass Spectrum) Displays the m/z and........
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Western Blotting 1. Experimental Principle Western Blotting is a technique used to detect the presence and expression levels of specific proteins in samples. The basic principle involves separating proteins using SDS-PAGE electrophoresis, transferring the proteins to a membrane (e.g., PVDF), and then detecting the target protein using specific antibodies. 2. Experimental Steps (1) Protein Sample Preparation: Extract proteins from cells or tissues. (2) SDS-PAGE Electrophoresis: Use electrophoresis to........
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• How to Perform Native Protein Electrophoresis
Native Polyacrylamide Gel Electrophoresis (Native PAGE) is a protein separation technique performed under non-denaturing conditions. The goal of Native PAGE is to separate proteins based on their charge, shape, and size while preserving their native structure and biological activity. The operation is relatively simple, and the steps are as follows: 1. Prepare Polyacrylamide Gel Choose the appropriate concentration of polyacrylamide gel, typically between 5-20%. Higher concentrations separate smaller........
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Gel Concentration Selection 1. The choice of gel concentration should be based on the molecular weight range of the protein to be tested. Generally, lower gel concentrations are suitable for larger proteins, while higher concentrations are better for smaller proteins. 2. Commonly used gel concentrations range from 8% to 15%. For larger proteins, a lower concentration such as 8% is recommended, while for smaller proteins, higher concentrations like 12% or 15% are ideal. 3. If the molecular weight of the.....
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• Protein Precipitation After Heating Denaturation in WB Experiments
In Western Blot (WB) experiments, protein precipitation after heating denaturation is a common issue. This is usually caused by the following factors: 1. High Protein Concentration If the protein concentration in the sample is too high, excessive aggregation and precipitation may occur during heating. 2. Inappropriate Sample Buffer The sample buffer used may not be suitable for the protein being analyzed. For example, improper pH or salt concentration of the buffer can affect protein solubility.
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Using the COG database for functional annotation analysis not only reveals the most abundant functional categories but also provides a detailed distribution of genes across various biological categories. Below is a method for writing a COG functional annotation analysis, which we hope will be helpful to you. 1. Introduction In the introduction, briefly introduce the significance of the COG database and its main uses. Explain the reasons for using the COG database to functionally annotate a specific genome..
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When developing quantitative methods in LC-MS, determining the quantitative and qualitative ion pairs is critical. Below are some guidelines on how to choose them: 1. Quantitative Ion Pairs Typically, the ions with the highest intensity are selected as quantitative ions because they offer the best signal-to-noise ratio. In practice, the full-scan mass spectrum of the target compound should first be obtained, and the highest abundance, representative ions should be identified. These ions can be molecular....
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Post-translational modifications (PTMs) of proteins in the cell are crucial because these modifications not only ensure that proteins have the proper conformation and function, but also regulate their localization and stability within the cell. PTMs provide a fine mechanism for regulating protein functions and activities, and are involved in controlling various biological processes. Why Proteins Need Post-Translational Modifications 1. Functional Diversity Modifications increase the functional diversity....
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The IOD (Integrated Optical Density) value, or integrated optical density, is the integral of the optical density within a specific region, generally used to analyze the color or grayscale distribution in an image. In SDS-PAGE protein purity analysis, it can be used to measure the intensity of specific protein bands, and this intensity is typically proportional to the protein content. In SDS-PAGE analysis, we generally do not directly measure the IOD value. Instead, we assess the relative abundance of......
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• What Causes Bromophenol Blue Diffusion in the Concentrating Gel During WB Protein Electrophoresis
Bromophenol blue is a commonly used tracking dye in SDS-PAGE electrophoresis to monitor the migration of samples. Its migration speed is generally similar to that of small molecular weight proteins, making it useful for observing the electrophoresis process. Causes of Diffusion Phenomenon 1. Gel Concentration If the concentration of the concentration gel is improperly set, it may lead to the diffusion of bromophenol blue. 2. Electrophoresis Conditions Voltage, current, buffer pH, and composition can all....
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