Western Blotting Protocol and FAQs

Western blotting is a widely employed technique in both scientific research and clinical diagnostics, providing a reliable method for analyzing proteins in complex mixtures. It is primarily used for detecting specific proteins,as well as determining their relative abundance in various samples. By utilizing specific antibodies that bind to the target protein, Western blotting allows for the identification and quantification of proteins of interest within a heterogeneous sample. This technique is particularly valuable in the study of protein purification processes, enabling researchers to assess the purity and presence of proteins at different stages of purification. Additionally, Western blotting plays a crucial role in evaluating protein expression levels in cells under different experimental conditions, such as in response to drugs, stress, or disease. Its versatility has made it a cornerstone in many fields, including cell biology, molecular biology, biochemistry, and clinical diagnostics, where it is used to investigate the underlying molecular mechanisms of diseases, including cancer, neurodegenerative disorders, and immune-related conditions.

The history of Western blotting dates back to 1979, when British biologist Alistair J. Towbin and his colleagues first proposed and implemented this method in their research. Towbin et al. originally used electrophoresis to separate proteins and transfer them through a polyethylene membrane, and then performed specific detection through antibodies. This method has been widely used in cell biology and molecular biology research, and has become one of the gold standard techniques for protein analysis.

The Principle of Western Blotting

Protein Separation

First, proteins in the sample were separated by gel electrophoresis (usually SDS-PAGE, polyacrylamide gel electrophoresis) according to molecular size.

Protein Transfer

The separated protein is then transferred by electrophoresis to a solid support, usually a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose membrane.

Antibody Detection

On the transferred membrane, specific antibodies are used to recognize the target protein. This antibody is usually labeled (such as enzyme labeling) and can produce measurable signals (such as chemiluminescence).

Signal Detection

The signal of the antibody-antigen reaction is obtained by an appropriate detection system (such as a chemiluminescence imaging device) to achieve qualitative and quantitative analysis of the target protein.

Reagents And Instruments required

1 Electrophoresis buffer : used for protein separation in SDS-PAGE gel and electrophoresis system.
2 Blocking solution : such as skimmed milk powder or BSA, to prevent non-specific antibody binding.
3 Primary antibody and secondary antibody : The primary antibody is a specific antibody against the target protein, and the secondary antibody is generally an enzyme-labeled antibody bound to the primary antibody.
4 Substrate solution : such as chemiluminescence substrate or colorimetric substrate, used for signal detection of labeled antibody.
5 Protein calibrators : Protein standards such as pre-defined sizes are used to confirm the molecular weight of the protein in the sample.
6 SDS-PAGE gel electrophoresis system : for protein separation.
7 Transmembrane system : used for protein transfer from the gel to the membrane.
8 Chemiluminescence imaging system : used to detect the signal after antibody-antigen reaction.
9 Shaker : for incubating antibodies.
10 Micropipettor : for precise dispensing of reagents and samples.
11 Membrane : PVDF membrane or nitrocellulose membrane is usually used for protein transfer and antibody detection.

The Steps of Western Blotting

Sample Preparation

Cell lysis

Extraction of proteins from cells or tissues. The commonly used lysis buffer contains protease inhibitors and phosphatase inhibitors to avoid degradation of the sample during extraction.

Protein quantification

Protein concentration was determined by BCA method and Bradford method.

Electrophoresis Separation (SDS-PAGE)

Sample preparation

The sample was mixed with SDS sample buffer and heated at 95 ° C for 5-10 minutes to ensure complete denaturation of the protein.

Gel electrophoresis

The treated protein samples were loaded into SDS-PAGE gel, usually using polyacrylamide gel. The application of electric field makes the protein separate according to the molecular size, and the small molecular weight protein migrates faster.

Transfer Film

Protein transfer

The separated protein is transferred to a nitrocellulose membrane (NC membrane) or a polyvinylidene fluoride membrane (PVDF membrane) by electrotransfer.

Blocking

In order to reduce non-specific binding, a blocking solution containing non-fat emulsion (BSA or skim milk powder) was used to block the blank area on the membrane, usually for 1 h.

Antibody Incubation

Primary antibody incubation

The membrane was added with a solution containing a specific primary antibody (the main antibody against the target protein), usually incubated for 1 hour to overnight.

Washing

Wash the membrane with TBS-T (TBS with Tween-20) to remove unbound primary antibodies.

Incubation of secondary antibody

Incubation with a labeled secondary antibody (usually HRP or fluorescently labeled secondary antibody), the secondary antibody can bind to the primary antibody and amplify the signal.

Signal Detection

Chemiluminescence detection

If HRP-labeled secondary antibody is used, a chemiluminescence substrate (such as ECL) is added, and a chemiluminescence signal that can be detected by an exposure instrument is generated through a chemical reaction.

Fluorescence detection

If a fluorescently labeled secondary antibody is used, it is detected by a fluorescence imaging device.

Result Analysis

Imaging of protein bands

The signal captured by the imaging system will be displayed as a protein band, and the intensity of the band is related to the expression of the target protein.

Quantitative analysis

The intensity of the bands was analyzed by software, and the expression level of the target protein was calculated and compared with the internal reference protein (such as β-actin or GAPDH).

Figure 1. Schematic representation of the Western Blotting Procedure. (Reference source: Protein purification and analysis: next generation Western blotting techniques.）

FAQ

1. What is the difference between nitrocellulose and PVDF membranes?

Nitrocellulose membranes are easier to handle, have lower background noise, and are suitable for many applications. However, they have limited protein binding capacity. PVDF membranes have a higher protein binding capacity and are more durable, making them ideal for long-term storage of blotted proteins. They require activation with methanol before use.
2. How to solve the high background of Western blotting diagram?

High background signals in Western blotting are often caused by non-specific antibody binding or incomplete blocking. (1) Optimization of the blocking solution : Blocking is essential to prevent non-specific binding of antibodies to the membrane. Common blockers include skimmed milk powder, bovine serum albumin (BSA) or commercial blocking buffer. (2) Refine Washing Steps: Inadequate washing between steps can lead to residual antibody binding, increasing background. Ensure thorough washing with an appropriate washing buffer (usually TBST or PBST) to remove any unbound antibodies. Increase the number of washes or the wash duration if necessary, especially if high background persists. Using too many antibodies can lead to non-specific binding and lead to high background. Try to further dilute the primary or secondary antibody, or use a more specific antibody to lower the background.
3. Blurred or unclear stripes.

Blurred or unclear stripes on a Western blot can result from incomplete electrophoresis separation or insufficient membrane transfer. If the protein is not well separated during electrophoresis, fuzzy bands may appear. Adjust the running time, voltage, and gel concentration of the electrophoresis gel to achieve clear separation of proteins.Incomplete protein transfer from gel to membrane may lead to blurred or missing bands. Ensure that the appropriate membrane type (e.g. PVDF or nitrocellulose) is used and that the transfer conditions are optimal. Adjust the transfer voltage, time and buffer composition if necessary.
4. The signal is too weak when exposed.

(1) Increase antibody concentration : If the antibody concentration is too low, the signal may be too weak to detect correctly. Consider increasing the concentration of the first antibody or the second antibody, and perform antibody titration to find the optimal antibody concentration to enhance the binding to the target protein. (2) Ensure complete membrane transfer : Incomplete transfer of proteins from the gel to the membrane may also lead to weak or missing signals. After transfer, the membrane is examined using reversible protein staining to ensure the correct transfer of the protein. If the transmission efficiency is low, the conditions such as voltage, time, buffer composition and transmission equipment are optimized. (3) Prolong the exposure time : If the signal is too weak, balance the exposure time and signal clarity, and use different lengths of test exposure to find the best time to provide a good signal-to-noise ratio.

reference

[1] Sule R, Rivera G, Gomes AV. Western blotting (immunoblotting): history, theory, uses, protocol and problems. Biotechniques. 2023;75(3):99-114. doi:10.2144/btn-2022-0034

[2] Kurien, B. T., & Scofield, R. H. (2015). Western blotting: an introduction. Methods in molecular biology (Clifton, N.J.), 1312, 17–30. https://doi.org/10.1007/978-1-4939-2694-7_5

[3] Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics. 2017;14(11):1037-1053. doi:10.1080/14789450.2017.1388167

[4] Kurien BT, Scofield RH. Western blotting. Methods. 2006;38(4):283-293. doi:10.1016/j.ymeth.2005.11.007

[5] Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015;1318:87-96. doi:10.1007/978-1-4939-2742-5_9