Protein Analysis FAQ

• • Why Do Dual Bands Reappear After Antibody Stripping in Western Blot When Probing for Closely Migrating Targets?

  Western blotting is widely employed to assess the expression of specific proteins in biological samples. When two protein bands migrate closely on the membrane, a common strategy involves detecting one target first, followed by antibody stripping and reprobing for the second target. However, the reappearance of both bands during the second detection may be attributed to the following factors: Incomplete Antibody Stripping The antibody stripping procedure may not have fully removed the initial primary and...

• • Can WB Membranes Be Used for Mass Spectrometry?

  Yes, proteins immobilized on Western Blot (WB) membranes can be subjected to mass spectrometry analysis. During the Western Blot procedure, proteins are transferred onto PVDF or nitrocellulose membranes. These protein bands can subsequently be excised from the membrane, enzymatically digested, and analyzed by mass spectrometry to identify the proteins or to perform further characterization.

• • What to Do If TLR4 and NF-κB Bands on WB Are Reversed? Secondary Antibody Diluted, Is Actin Normal?

  Signal inversion (i.e., bands appearing lighter than the background) for TLR4 and NF-κB in Western blotting—despite appropriate secondary antibody dilution and normal actin control—may result from multiple underlying factors: Excessive Secondary Antibody Concentration If reducing the secondary antibody concentration fails to resolve the issue, further dilution or substitution with an alternative secondary antibody may be warranted to minimize background signal amplification. Overconcentration of Primary....

• • Why Do Stained Purified Proteins Show No Signal in Western Blot Detection Despite Proper Technique and Reagents?

  In Western blot (WB) analysis, if stained purified proteins yield visible bands but no signal is observed during immunodetection—despite ruling out procedural or reagent-related errors—several additional factors may be responsible: Limited Antibody Specificity The primary or secondary antibody may lack sufficient specificity for the target protein, resulting in failure to bind the relevant epitope. Using alternative antibodies, preferably those previously validated for Western blot, may resolve this issue..

• • Is It Problematic to Measure Only Phosphorylated Proteins Without Total Levels in WB Pathway Analysis?

  Whether the omission of total protein detection poses a significant issue depends on the goals of the study and the design of the experiment. In general, the failure to assess both total protein levels and phosphorylation status can affect the accuracy and interpretability of the results in several important ways: Baseline Protein Expression Levels Quantifying total protein provides an essential internal control to establish the baseline expression of the target protein within the sample. Without this infor

• • How to Distinguish Mixed WB Protein Samples?

  To differentiate Western Blot (WB) protein samples that have been accidentally mixed or cross-contaminated, multiple experimental techniques are typically required. Once samples are compromised, distinguishing them based solely on visual inspection or basic physical characteristics becomes highly challenging. The following approaches may be employed: Repetition of Western Blot and Comparative Analysis If original WB data or electrophoresis images are still available, repeating the WB experiment and ........

• • What Are the Underlying Principles of the Western Blot Technique?

  Western blotting, also referred to as immunoblotting, is a widely used analytical technique for detecting the presence and quantifying the abundance of specific proteins in biological samples. The methodology is grounded in several key principles derived from biochemistry and molecular biology: Electrophoretic Separation of Proteins Proteins generally carry a net negative charge under alkaline pH conditions. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the detergent SDS is used..

• • Is There a Terminological Distinction Between “Protein” and “Proteins”?

  The terms protein and proteins are frequently used interchangeably, both denoting macromolecular compounds composed of amino acids. These molecules perform a wide range of biological functions within cells, such as catalyzing biochemical reactions (as enzymes), acting as signaling molecules, providing structural support, and participating in immune responses. Protein This term typically refers to a specific and well-characterized protein molecule or type. Examples include hemoglobin and antibodies.

• • What Are PLS-DA/OPLS-DA 2D Plots Used For?

  PLS-DA (Partial Least Squares Discriminant Analysis) and OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) are widely employed multivariate statistical methods, particularly in high-throughput biomedical research areas such as metabolomics and proteomics. These approaches enable the reduction of high-dimensional data into two- or three-dimensional representations, facilitating the visualization of inter-sample variation. PLS-DA is grounded in partial least squares regression and is primarily.

• • How Can RMSECV Values Be Accessed and Interpreted After PLS-DA/OPLS-DA Model Construction?

  Upon completing PLS-DA (Partial Least Squares Discriminant Analysis) or OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) modeling and generating the corresponding 2D plots, the model’s performance can be assessed using specialized software platforms such as SIMCA or MetaboAnalyst, among others. In SIMCA, the Root Mean Square Error of Cross-Validation (RMSECV) can be located in the "Results" panel on the left-hand side of the interface. This metric quantifies the average predictive error.....