Protein Analysis FAQ

• • How Are Extracted Peptides from Analytical Samples Processed?

  The extraction of peptides from analytical samples generally comprises several key steps, which may vary depending on the sample type (e.g., cells, tissues, body fluids) and the intended purpose (e.g., mass spectrometry analysis, bioactivity assays). A representative peptide extraction workflow is outlined below:   1. Sample Preparation For solid samples (e.g., tissues or food matrices), preliminary physical disruption is typically required, such as by using a mortar and pestle, a tissue homogenizer, ......

• • Is There a Association Between SDS-PAGE Band Smearing and Protein Glycosylation?

  Yes, the diffusion or smearing of SDS–PAGE bands can be potentially associated with protein glycosylation. Glycosylation can introduce heterogeneity into protein samples, which refers to the occurrence of identical proteins in multiple glycoform variants. Such structural variability may result in the appearance of multiple discrete bands or diffuse bands during SDS–PAGE analysis. Furthermore, glycosylation can alter the interaction between proteins and sodium dodecyl sulfate (SDS), thereby affecting t......

• • How to Find New Cellular Pathways?

  The identification of novel cellular pathways is a complex endeavor that typically requires a multidisciplinary approach, integrating expertise from biology, chemistry, bioinformatics, and related fields. Presented below are widely adopted strategies and methodologies for the exploration and characterization of previously unrecognized cellular pathways:   1. Genomics and Transcriptomics Whole-genome sequencing and transcriptome profiling (RNA-seq) can be employed to detect previously uncharacterized g......

• • How to Perform Protein Quantification for Western Blot Analysis of White Adipose Tissue?

  First, the white adipose tissue should be homogenized and lysed to extract total proteins. RIPA buffer is a commonly used lysis reagent, as it effectively disrupts cellular membranes and releases intracellular proteins. Protease and phosphatase inhibitors must be added to the lysis buffer to prevent protein degradation during extraction.   Following protein extraction, quantification can be performed using a BCA protein assay kit. An appropriate volume of protein sample and standard is mixed with the ......

• • What Is the Most Critical Step in a Western Blot Experiment?

  Western blotting comprises multiple essential and interdependent steps, each of which plays a pivotal role in the accuracy and reliability of the results. Nevertheless, if one were to identify the most critical steps, the following could be considered:   1. Preparation of Protein Samples The foremost step is to ensure the integrity and concentration of proteins in the sample, as failure to do so may result in the inability to detect the target protein in subsequent stages. This requires the use of app......

• • What Methods Are Available for Determining the Connectivity of Multiple Disulfide Bonds in Polypeptides?

  Determining the connectivity of multiple disulfide bonds in a polypeptide is often challenging, particularly when multiple potential disulfide bridge arrangements are possible. Presented below are several commonly employed experimental and computational strategies for elucidating disulfide bond connectivity in polypeptides:   X-ray Crystallography 1. Principle X-ray diffraction enables direct visualization of the atomic arrangement within a protein, thereby allowing unambiguous determination of disulf......

• • Why Does GFP Show Two Bands on SDS-PAGE After FPLC, and How Can It Be Purified More Precisely?

  When green fluorescent protein (GFP) exhibits two distinct bands on SDS-PAGE after purification via fast protein liquid chromatography (FPLC), several factors may account for this phenomenon:   1. Partial Protein Degradation Proteolytic cleavage during expression, extraction, or storage may generate protein fragments with lower molecular weights than the intact GFP, resulting in additional bands.   2. Post-Translational Modifications Modifications such as phosphorylation or glycosylation can alter the......

• • Q&A of Label-Free Quantification Proteomics

  Q1: What are the key features of Label-Free Quantification (LFQ)? A1: 1. No chemical or isotopic labeling required Quantitation is derived directly from MS signal intensity, avoiding the extra steps and cost introduced by labels.

• • In WB, Should the Sample With the Lowest Gray Value Be Diluted for Equal Loading and Mixed With the Calculated Diluent?

  The meaning of the dilution ratio calculated from the sample gray value is how many times the original sample needs to be diluted, rather than directly adding a volume of diluent equal to the ratio. Below is a procedure for diluting the sample according to the calculated dilution ratio: Steps 1. Determine the target gray value: Select a target gray value, which is usually the one with the lowest gray value among the samples as the reference. 2. Calculate the dilution ratio: For each sample, calculate the...

• • What Preparations Are Required for Conducting a Western Blot Experiment?

  Prior to conducting a Western Blot (WB) experiment, a series of essential preparations should be carried out to ensure the reliability and reproducibility of the results: Experimental Design 1. Identify the target protein and select appropriate primary and secondary antibodies. 2. Choose a suitable internal loading control (e.g., GAPDH or β-actin). 3. Define the sample type (e.g., cell lysates, tissue extracts). Sample Preparation 1. Collect and process biological samples (e.g., cultured cells, tissues).