Western Blot

SDS-PAGE and Western Blot

Western blotting is a well-established molecular biological method for the detection, analysis and quantification of proteins [1]. This is done by transferring them onto a carrier membrane, which is called “blotting” [2]. Subsequently, proteins can be visualized by immunodetection [3]. The name is inspired by the Southern blot. Edwin Southern, who is considered the inventor of the blotting technique, has developed the first blot procedure for the detection of DNA fragments in 1975. As a name game, the detection of RNA was then named Northern Blot, while the detection for proteins was called Western Blot. Other methods include the Southwestern blot for detecting DNA-protein interactions and the Northwestern blot for detecting RNA-protein interactions [2].

Principle_WB_EN

Workflow of Western blotting. In the first step, the protein lysate to be analyzed is separated by gel electrophoresis (1). The proteins are then transferred to a membrane (2) and analyzed by immunodetection. In this process, a primary antibody binds to the protein of interest and an enzyme-coupled secondary antibody binds to the primary antibody. After addition of the appropriate substrate, a signal can be detected (3) and the proteins are visualized as bands on the membrane (4) (figure created with biorender.com).

Before the actual Western blot can be performed, the protein lysate to be analyzed must first be separated. This can be achieved by different methods, for example by means of an SDS-PAGE, a native PAGE, isoelectric focusing or a 2D gel electrophoresis. Depending on the method chosen, the proteins are separated on the basis of their size, charge or spatial structure [2]. In SDS-PAGE (polyacrylamid gel electrophoresis), for example, separation is based on molecule size: sodium dodecyl sulfate (SDS) is added to the sample material, which attaches itself to the proteins, denaturing them. This gives them a strong negative charge, which masks their intrinsic charge. Since the negative charge is proportional to the mass of the protein, its migration speed in the gel depends only on its molecular size [4].

In the next step, the separated proteins are transferred to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane using another electric field that is perpendicular to the gel. The protein bands, now tightly bound on the membrane, can be detected by various methods, the most commonly chosen being immunodetection. For this purpose, the free binding sites on the membrane are blocked first. This is done by applying proteins that cannot be recognized by the antibodies used. Often the membrane is treated with bovine serum albumin, milk powder solution or gelatin. After the blocking step, specific mono- or polyclonal primary antibodies are added to the membrane, which bind to the protein of interest. Subsequently, enzyme-coupled (e.g. HRP) secondary antibodies are used and bind to the primary antibodies. After multiple washes to remove excess antibodies, a substrate is added which is converted by the enzyme and allows detection of the protein of interest [5].

The Western blot is one of the most widely used protein analytical methods [6]. It is employed in biochemical and medical research as well as in diagnostics. It is often used to detect disease-relevant proteins, especially specific antibodies in blood serum for the diagnosis of infections. The pattern of the protein bands produced by blotting can provide clues to the time of infection and help distinguish between different pathogens [4]. However, the Western blot can also be used for semi-quantitative analyses or for qualitative detection of protein transformations, such as post-translational modifications [1].

Are you looking for the right reagents or an efficient kit for your next Western blot? Then you will certainly find what you are looking for in our extensive range! You can see a small selection of suitable products here. Below the table you will also find all antibodies from our portfolio that you can use in the Western Blot.

Product Name	Product Type	Product Number
Anti-Red Fluorescent Protein (RFP)	Primary Antibody	600-401-379
Anti-Green Fluorescent Protein (GFP)	Primary Antibody	600-101-215
Anti-NLRP3/NALP3, clone Cryo-2	Primary Antibody	AG-20B-0014-C100
Anti-Caspase-1 (p20) (mouse), clone Casper-1	Primary Antibody	AG-20B-0042-C100
Anti-phospho-RPA32 (Ser4/Ser8)	Primary Antibody	A300-245A
Revitablot(TM) Western Blot Stripping Buffer	Buffer	MB-085-0500
Mouse TrueBlot® Western Blot Kit	Toolkit	88-8887-31
Rabbit TrueBlot® Western Blot Kit	Toolkit	88-8886-31
Goat TrueBlot® Western Blot Kit	Toolkit	88-8884-31
TrueBlot® Immunoprecipitation and Western Blot Kit for GFP Epitope Tag	Toolkit	88-8887-215
TrueBlot® Immunoprecipitation and Western Blot Kit for 6X HIS Epitope Tag	Toolkit	88-8887-382
Western Blot Detection Kit	Toolkit	E-IR-R304A.50
High Accuracy and Absorbability Western Blot Detection Kit	Toolkit	E-IR-R304B.50
METTL3, FLAG-Tag (positive control)	Rec. Protein	BPS-100055
BCL2A1, His-Tag (positive control)	Rec. Protein	BPS-100077

Primary Antibodies for Western Blot at Biomol

Secondary Antibodies for Western Blot at Biomol

Sources

[1] https://www.uniklinikum-saarland.de/de/einrichtungen/fachrichtungen/zellbiologie/seminar_zellbiologie_20192020/blots/anwendungsbeispiele, 10.06.2023

[2] https://de.wikipedia.org/wiki/Western_Blot, 24.01.2023

[3] A. M. Gressner, O. A. Gressner: Western blot. In: Lexikon der Medizinischen Laboratoriumsdiagnostik. Springer Berlin Heidelberg. S. 2505–2506 (2019).

[4] https://flexikon.doccheck.com/de/SDS-PAGE, 30.01.2023

[5] https://flexikon.doccheck.com/de/Western_Blot, 24.01.2023

[6] C. P. Moritz. 40 years Western blotting: A scientific birthday toast. Journal of Proteomics (2020).