Protein Analysis FAQ

• • 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).

• • Does the Presence of Ice During WB Protein Extraction Influence Experimental Outcomes?

  If ice from the storage container is inadvertently introduced into the protein extraction process, it may alter both the concentration and the purity of the protein sample: Dilution Effect Upon melting, the ice increases the total liquid volume, thereby diluting the sample and potentially reducing the protein concentration. Sample Contamination Although uncommon, ice produced under non-sterile conditions may serve as a source of microbial or other extraneous contaminants. For most Western Blot (WB) .......

• • Is Reuse of Electrophoresis Buffer in Western Blotting Acceptable?

  In most cases, reusing electrophoresis buffer in Western blot experiments is not recommended. The buffer components—such as Tris, SDS, and other ionic species—may become depleted or accumulate contaminants, including residual proteins, cellular debris, and other impurities introduced during electrophoresis. These changes can compromise the consistency and accuracy of protein migration and ultimately affect the reliability of experimental outcomes. Furthermore, previously used running buffer carries a risk..

• • What Is the Purpose of Keeping Tissue Homogenate on Ice During Total Protein Extraction in Western Blot?

  In Western blot experiments, briefly placing the tissue homogenate on ice following homogenization serves a critical role in preserving protein integrity. This step primarily aims to prevent thermal denaturation of proteins caused by heat generated during mechanical disruption. Maintaining the homogenate at low temperatures helps stabilize the native conformation of proteins and minimizes the activity of endogenous proteases and phosphatases, thereby improving the accuracy and reproducibility of downstream

• • Is Yellow Coloration of Intestinal Tissue Lysate Supernatant Normal During Western Blot Sample Preparation?

  In the context of Western blotting, it is common for the supernatant obtained from intestinal tissue protein extraction to exhibit a yellow coloration. This is typically due to the presence of bile pigments naturally found in intestinal tissue, which can impart a yellow hue to the lysate. Importantly, the color of the lysate does not directly influence Western Blot outcomes. Instead, the reliability of the experiment primarily depends on the efficiency of protein extraction and the accuracy of concentration