Protein Level Detection

Reduced levels of target proteins are an important reference for evaluating the effects of RNA interference. Here, BOC Sciences provides Western blot-based protein knockdown testing services to assist clients in efficiently evaluating the effects of gene silencing.

What is Western Blot Analysis?

Western Blot, also known as protein blotting, is a protein detection technique widely used in biology. western blot uses polyacrylamide gel electrophoresis with antibodies as "probes" and labeled secondary antibodies to "develop the color" of the protein being detected. Western Blot uses polyacrylamide gel electrophoresis, using antibodies as "probes" and labeled secondary antibodies for "color development" to detect proteins specifically and efficiently. Protein samples separated by PAGE (polyacrylamide gel electrophoresis) are transferred to a solid-phase carrier (e.g., nitrocellulose film), which adsorbs proteins in the form of non-covalent bonds and maintains the type of peptide separated by electrophoresis and its biological activity. The protein or peptide on the solid-phase carrier is used as an antigen, which is immunoreacted with the corresponding antibody, and then reacted with an enzyme- or isotope-labeled secondary antibody, which is used to detect the protein components specific to the expression of the target gene separated by electrophoresis through substrate chromatography or autoradiography.

Figure 1. Western blot analysis.

Protein Level Detection Services from BOC Sciences

• RNAi design and synthesis

BOC Sciences' design and synthesis services for RNAi molecules corresponding to the coding sequences of target proteins, such as small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs.) BOC Sciences ensures that the RNAi sequences specifically interact with the mRNAs of the target proteins.

• RNAi Transfection

BOC Sciences provides RNAi molecular transfection optimization services, achieved through a variety of transfection methods, such as chemical transfection, electroporation, or viral transfection. BOC Sciences ensures that the RNAi is able to enter the cell and target the mRNA of the target protein.

• Cell Lysis, Protein Separation, Membrane Transfer

BOC Sciences uses SDS-PAGE electrophoresis to separate out proteins from cell lysates, and then transfers the separated proteins from the gel to a polymer membrane (e.g., PVDF or NC membrane).

• Immunodetection

BOC Sciences incubates transmembrane-transformed proteins with a primary antibody specific to the target protein (primary antibody), followed by incubation using a secondary antibody that is different from the primary antibody's species of origin. The secondary antibody is usually conjugated to an enzyme or a fluorescent marker to produce a detection signal. This detection signal is the primary basis for assessing the effect of RNAi's interference with the target protein.

Figure 2. BOC Sciences' protein level detection procedure.

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