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Western Blot (WB)


Western blot (WB) is a core technique in cell and molecular biology, which is used to detect the presence of a specific protein in a complex mixture extracted from cells. The WB procedure relies upon three key elements to accomplish this task: the separation of protein mixtures by size using gel electrophoresis, the efficient transfer of separated proteins to a solid support, and the specific detection of a target protein by appropriately matched antibodies. Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or an imaging system. Our WB protocol involves the following 5 main steps: sample preparation, SDS-PAGE gel electrophoresis, protein transfer, immunoblotting, detection. Creative Biolabs provides very detailed steps instruction for each one. Please refer to Western Blot Protocols & Troubleshooting & Guide for more information.

Detection Method

Detailed procedures for detection of a WB may vary widely. One common variation involves direct vs. indirect detection. The following diagram and tables show the specific differences between them.

Detection Method

Table 1. Direct Detection Method

Advantages Disadvantages
  • It is a quick methodology because only one antibody is used
  • Immunoreactivity of the primary antibody may be reduced as a result of labeling
  • Cross-reactivity of secondary antibody is eliminated
  • Labeling a primary antibody for each target protein is time-consuming and expensive
  • Double probing is easily achieved using different labels on primary antibodies from the same host
  • There is no flexibility in choice of primary antibody label from one experiment to another
  • Minimal signal amplification

Table 2. Indirect Detection Method

Advantages Disadvantages
  • Sensitivity is increased because each primary antibody contains several epitopes bound by the labeled secondary antibody, which amplifies the signal
  • Cross-reactivity may occur with the secondary antibody, resulting in nonspecific binding
  • A wide variety of labeled secondary antibodies are available commercially
  • An extra incubation step is required in the procedure
  • Because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection, it is versatile
  • Immunoreactivity of the primary antibody is maintained because it is not labeled
  • Different detection markers can be used with the same primary antibody

How to Choose Appropriate Secondary Antibody?

How to Choose Appropriate Secondary Antibody?

Protein Electrophoresis

Protein electrophoresis is a standard laboratory technique by which charged protein molecules are transported through a solvent by an electrical field. Most biological molecules carry a net charge at any pH other than their isoelectric point and will migrate at a rate proportional to their charge density. Denaturing and reducing SDS-PAGE with a discontinuous buffer system is the most widely used electrophoresis technique and separates proteins primarily by mass. Nondenaturing PAGE (also known as native-PAGE) separates proteins according to their mass/charge ratio. Two-dimensional (2D) PAGE separates proteins by native isoelectric point in the first dimension and by mass in the second dimension.

SDS-PAGE vs. Native-PAGE

Recent Progresses

Although WB is a relatively mature technology, there are still researchers working on it to upgrade this technique. The development of improved detection methods can expand multiplexing capabilities and push detection limits. Progress in these areas will likely continue at a rapid pace as improved method for microfluidics, microscale analysis systems, and label-free biosensing continue to advance the field.

Examples of microfluidic design and on-capillary protein immobilization approaches. Fig.1 Examples of microfluidic design and on-capillary protein immobilization approaches. (Sanders, 2016)

More WB Resource

Western Blot Protocols & Troubleshooting & Guide

Western Blot Protocol

Western Blot Troubleshooting

Sample Preparation in Western Blot Assay

Protein Transfer from Gel to Membrane in Western Blot Assay

Running an SDS-PAGE Gel in Western Blot Assay

Western Blot Illustrated Assay

Stripping and Reprobing Protocol

Western Blot Storage Protocol


  1. Sanders, B. J.; et al. Recent advances in microscale western blotting. Analytical methods. 2016, 8(39): 7002-7013.