Protein Analysis FAQ

• • How Can a Protein Be Identified as a Homodimer (a Dimer With Identical Subunits)

  Determining whether a protein forms a homodimer (a dimer consisting of identical subunits) can be assessed using multiple approaches:   Mass Spectrometry 1. Principle Mass spectrometry allows precise determination of the molecular mass of proteins or peptides.   2. Procedure Perform mass spectrometry under denaturing conditions to measure the molecular mass of individual subunits. Conduct mass spectrometry under native conditions to determine the mass of the intact protein complex, thereby confirming ......

• • What Could Cause a Western Blot Band to Appear Blank and Overexposed?

  In a Western Blot (WB) experiment, bands that appear blank or overexposed (typically due to excessive exposure during imaging) may be caused by the following factors:   1. High Protein Concentration in Samples A high protein concentration in the loaded sample can lead to excessive signal intensity, causing the bands to appear overexposed. To resolve this, the sample should be diluted, and the loading volume reduced.   2. Excessive Primary or Secondary Antibody Concentration High concentrations of anti......

• • What Causes the Loss of Protein Bands and Markers During Western Blot?

  The loss of protein bands and molecular weight markers during Western Blot may result from several factors:   1. Insufficient Protein Loading A low protein loading amount may lead to weak or undetectable bands. Insufficient protein in the sample can also reduce transfer efficiency, resulting in faint or missing bands.   2. Excessive Electrophoresis Duration Overly long electrophoresis can cause small proteins to migrate off the gel, preventing their subsequent transfer to the membrane. It is essential......

• • Can the Protein Stock Be Boiled Before Adding Loading Buffer in Western Blotting? What if It Has Already Been Boiled?

  In Western blot (WB) experiments, protein samples are typically mixed with loading buffer before undergoing heat-induced denaturation. Boiling the protein stock solution before adding the loading buffer may cause excessive denaturation and protein aggregation, potentially compromising WB results. Therefore, it is advisable to store the protein sample at low temperatures before mixing it with the loading buffer and to use it in subsequent WB experiments.   If the protein stock has been mistakenly boile......

• • Does the Protein Degrade if Mixed with Loading Buffer for WB and Frozen at -80°C Without Prior Boiling for Denaturation?

  In Western blot (WB) experiments, when protein is mixed with loading buffer but not boiled for denaturation, and is subsequently frozen at -80°C, protein degradation generally does not occur.   The loading buffer typically contains reducing agents (e.g., β-mercaptoethanol) and SDS (sodium dodecyl sulfate), which facilitate protein denaturation and dissociation. During WB experiments, protein samples are typically mixed with loading buffer and then boiled to denature the proteins and dissociate them in......

• • Does the Appearance of Two Bands in a Western Blot Suggest Protein Degradation?

  The presence of two bands in a Western blot may indicate protein degradation. However, other factors could also account for this phenomenon, including:   1. Protein Degradation Protein degradation may occur during sample handling, storage, or experimental procedures, leading to the formation of multiple bands. To minimize degradation, protease inhibitors should be used, and repeated freeze-thaw cycles should be avoided.   2. Post-Translational Modifications Post-translational modifications (e.g., phos......

• • How Can Glycosylation Sites and the Molecular Weight After Glycosylation Be Analyzed, and Which Prediction Tools Are Available?

  Methods for Analyzing Glycosylation Sites 1. Experimental Approaches (1) Mass Spectrometry (MS) Analysis: Mass spectrometry (MS) is the gold standard for identifying glycosylation sites in proteins. MS/MS analysis of specific peptide fragments enables precise localization of glycosylation sites. (2) Immunoblotting: Detection of glycosylated proteins can be achieved using antibodies specific to glycosylation sites.   2. Bioinformatics-Based Prediction (1) Prediction Tools: Computational tools such as N......

• • Which Amino Acids Can Be Ubiquitinated?

  Ubiquitination is a post-translational modification in which ubiquitin, a small regulatory protein, is covalently attached to substrate proteins. This modification predominantly occurs on specific amino acid residues:   1. Lysine (K) The ε-amino group of lysine is the primary site for ubiquitination. Ubiquitin’s C-terminal glycine forms a covalent isopeptide bond with the lysine residue of the substrate protein.   2. Cysteine (C) Although less common, cysteine residues can also undergo ubiquitination.......

• • What Buffer Should Be Used to Dilute Boiled Protein Lysate for Western Blotting?

  For Western blotting, boiled protein lysate can be diluted using one of the following buffers:   Protein Extraction Buffer The extraction buffer used for initial protein isolation (e.g., RIPA buffer, PBS, or Tris buffer) can also be used for dilution, helping to maintain protein stability.   1X Loading Buffer A 1X SDS-PAGE loading buffer, typically composed of SDS, β-mercaptoethanol, glycerol, bromophenol blue, and Tris-HCl, ensures proper protein migration during electrophoresis.   Dilution Procedure......

• • Why Is the Recommended Protein Load for Western Blot 20–30 μg? How Does Loading Affect Results? Does It Depend on the Purpose?

  In Western Blot (WB) experiments, the recommended protein loading amount typically ranges from 20 μg to 30 μg. This range is generally sufficient to produce well-defined bands while preventing overloading, which may cause signal saturation or band smearing. Excessive protein loading can lead to overly intense signals, blurred bands, or even band aggregation. Conversely, insufficient protein loading may result in weak signals, making it challenging to detect the target protein.   Moreover, the optimal ......