Protein Immunoblotting and Electrotransfer Services

    The process where proteins separated by electrophoresis are transferred or “blotted” from the gel matrix onto a membrane (usually nitrocellulose or polyvinylidene difluoride (PVDF)), followed by subsequent antibody-based detection on the membrane surface, is known as western blot (WB) or immunoblotting. Mtoz Biolabs provides WB analysis services. Through highly selective and sensitive antibody-antigen interactions, WB can be used to detect specific target proteins from complex protein mixtures, such as tissue homogenates or cell extracts. The obtained data can be used for qualitative and semi-quantitative analysis of target proteins.

     

    Analysis Workflow

    WB is a widely used protein analysis technique for detecting specific proteins in tissue homogenates or extract samples. It uses gel electrophoresis to separate native proteins and denatured proteins by their 3D structures and peptide chain lengths, respectively. After separation, the proteins are transferred onto a membrane, where specific antibodies recognize the target proteins specifically. WB is widely applied in molecular biology, biochemistry, immunogenetics, and other molecular biology fields.

     

    In WB analysis, proteins are first separated from gel electrophoresis by various methods such as SDS-PAGE and IEF, by isoelectric point (pI), molecular weight, charge, or a combination of these factors. SDS-PAGE is commonly used for separation because all proteins are dissolved within the gel and migrate in the same direction, and the denaturing effect of SDS makes antigenic epitopes more easily recognizable.

     

    After gel electrophoresis analysis, proteins are transferred from the gel to a membrane made of nitrocellulose or PVDF for antibody detection. PVDF membranes have higher protein binding capacity than nitrocellulose membranes, but nitrocellulose membranes are better at binding smaller proteins. The main method of transferring proteins is electroblotting, which uses electricity to pull proteins from the gel into the PVDF or nitrocellulose membrane. Electrophoretic transfer methods include wet transfer, semi-dry transfer, and semi-wet transfer. If protein separation is done by IEF, then using pressure transfer diffusion to transfer proteins is more effective.

     

    The transfer process varies from one hour (semi-dry transfer) to overnight transfer (wet transfer). After the transfer is complete, the free binding sites on the membrane are blocked by the protein mixture, thus subsequent antibody detection is not interfered. The proteins transferred to the membrane can then undergo antibody detection. Membranes containing proteins are first incubated with primary antibodies, followed by detection with secondary antibodies that recognize the primary antibodies. Secondary antibodies generally carry a reporter group and have high sensitivity.

     

    The most sensitive detection method is using enhanced chemiluminescence (ECL): recognizing primary antibodies with an antibody-horseradish conjugate. Using special variants of ECL, protein bands as low as 1 pg can be detected.

     

    WB can also be used to track the phosphorylation of proteins, with antibodies that bind to all subtypes of phosphorylated proteins presenting a “bead-like” appearance on 2D gels. It can also be used to identify known and unknown proteins in complexes produced by IP, as long as the target proteins are in the gel, they can be identified by coomassie brilliant blue staining, cutting out the corresponding spots from the gel, and subsequently identifying them by mass spectrometry.

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