Protein Analysis FAQ
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• The Main Software Used for Predicting Post-Translational Modifications of Protein Sequences
The prediction of post-translational modifications (PTMs) in protein sequences is of great importance in bioinformatics and systems biology, as PTMs play a crucial role in regulating protein function, localization, and interactions. Several computational tools have been developed to predict PTMs, with some of the most commonly used ones listed below: 1. PhosphoSitePlus A comprehensive database providing information on experimentally identified post-translational modifications, particularly in disease-......
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• Can I Reboil My WB Sample After Storing It in the Fridge
Yes. If the sample has undergone prior boiling and has been stored at 4°C, it is recommended to reheat it before use to ensure that the components in the loading buffer, such as SDS and DTT (or β-mercaptoethanol), function effectively, allowing complete protein denaturation. Before reheating, ensure that the sample is fully thawed and gently mixed to prevent phase separation or precipitation of its components, which could affect experimental results.
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• Western Blot: Why Did This Situation Occur Where the Protein Ran onto the Marker
When proteins run onto the marker during a Western blot experiment, several factors may contribute to this phenomenon: 1. Excessive Sample Loading Loading an excessive amount of sample can cause proteins to migrate beyond the resolving capacity of the gel, resulting in some proteins running onto the marker. This typically occurs when the protein concentration is too high or when the sample volume is not accurately controlled during loading. Solution: Determine the loading volume based on both the protein...
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• Why Did Bands Appear in the WB Exposure? Are There Any Solutions
Overexposure in Western blot is typically caused by prolonged exposure time or a high antibody concentration. Potential solutions to mitigate this issue include: 1. Adjusting Exposure Time Shorten the exposure time, particularly when using chemiluminescent detection. 2. Optimizing Antibody Concentration Reduce the concentration of the primary or secondary antibody. 3. Washing Steps Increase the frequency or extend the duration of membrane washing to minimize nonspecific binding. 4. Blocking Use an adequ....
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• How to Interpret the Results of a Pull-Down Experiment
Pull-down experiments are widely used to study protein-protein interactions. In these experiments, specific antibodies or affinity reagents are employed to capture the target protein. Subsequently, protein analysis techniques are applied to detect and identify other proteins that interact with the target protein. Below, we outline key steps and considerations for interpreting pull-down experiment results. 1. Verifying Experimental Conditions Before analyzing pull-down experiment results, it is crucial to...
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• How to Analyze and Understand Post-Translational Modification of Proteins
In protein research, beyond analyzing their fundamental amino acid sequences, it is crucial to consider post-translational modifications (PTMs). PTMs refer to the chemical alterations proteins undergo after synthesis, which can significantly impact their structure and function. These modifications play a vital role in regulating protein stability, subcellular localization, biomolecular interactions, and activity. The types, detection methods, and functional significance of PTMs are outlined below........
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• Why Does Alix Protein Antibody Show Two Bands in WB? Which One Is Correct
Several factors may contribute to the appearance of two bands in Western Blot (WB) when using an Alix protein antibody: 1. Protein Isoforms Alix protein may exist in different isoforms, which differ slightly in molecular weight, leading to distinct bands on the gel. 2. Protein Degradation Products Partial degradation of Alix protein during cell lysis may result in the formation of smaller degradation fragments. These fragments can also be recognized by the same antibody, appearing as lower molecular........
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• Overview of Histone Modifications, Sites, and Their Significance
Histone modifications constitute a post-translational regulatory mechanism that profoundly influences chromatin structure and function. These modifications predominantly occur on the N-terminal tails of histones and include acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation, each playing a distinct role in gene regulation and genome stability. Below, we summarize the major types of histone modifications, their target sites, and their biological significance. Acetylation 1. Sites.....
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High-Resolution Mass Spectrometry (HRMS) is a powerful analytical technique that enables precise measurement of molecular masses, making it highly valuable for molecular weight identification. Deconvolution analysis is a widely used data processing approach in HRMS that enhances spectral resolution and improves signal-to-noise ratio. By applying appropriate deconvolution techniques, researchers can achieve more accurate molecular weight determinations, thereby increasing the reliability of compound ........
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• How to Pretreat Solids for GC-MS? Besides Internal Standard, Is NaCl Needed
Before performing gas chromatography-mass spectrometry (GC-MS) analysis, solid samples must undergo appropriate pretreatment to extract and concentrate target analytes while eliminating potential interfering substances. The standard pretreatment steps are as follows: 1. Grinding The solid sample should be ground to a suitable particle size using an appropriate method, such as a mortar and pestle or a mechanical homogenizer. Proper grinding enhances sample homogeneity and improves extraction efficiency.
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