Western blotting

Western blotting (WB) is a very common technique for the detection of a specific protein in a sample, for example a cell extract. The proteins within the sample are denatured and separated by SDS-PAGE, before they are transferred (blotted) to a nitrocellulose or PVDF membrane. In most cases, antibodies or antibody fragments are used for the detection of the protein of interest on the membrane. Usually, an unlabeled primary antibody recognizing the protein of interest on the membrane is detected by a labeled secondary antibody. Alternatively, the protein can also be detected directly with a labeled primary antibody. Various labels are used for detection, e.g. fluorescent dyes or the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), which catalyze a color or luminescence reaction. To minimize unspecific signal, the membrane is blocked with buffer containing milk powder or other proteins. After the antibody incubation steps, extensive washing of the membrane is required to remove unbound antibody.

Dot blotting

For dot blotting, protein samples are directly spotted onto a PVDF or nitrocellulose membrane. The membrane is then probed with a specific antibody or antibody fragment. In contrast to Western blotting, the samples are not separated by SDS-PAGE before they are applied to the membrane.
Dot blotting is a fast and effective method for probing multiple samples at the same time, e.g. different antibodies on the same antigen or the same antibody on multiple protein samples. As no information about the size of the recognized antigen is given, dot blotting is not suited for the discrimination of different protein variants such as protein fragments or proteins carrying post-translational modifications (PTMs), e.g. glycosylation, phosphorylation, etc.

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Staining with secondary antibodies

Primary antibodies bind to a specific target protein on the Western blot membrane. They are raised in animal species like rabbit, mouse, or chicken etc. Secondary antibodies are used for the detection of primary antibodies They recognize conserved, species specific regions of the primary antibody. For multiplexing with two or more primary antibodies from different species, it is important that the secondary antibodies selectively recognize their primary antibody and do not cross-react with immunoglobulins from other species. For detection in Western blotting, secondary antibodies are usually conjugated to the enzymes HRP or AP. Alternatively, the secondary antibodies may be conjugated to fluorescent dyes for read-out.

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Staining with Nano-Secondaries

What is a Nano-Secondary?

ChromoTek’s Nano-Secondaries are alpaca Nanobodies/ VHHs that bind to primary antibodies with high affinity in a species and subclass specific manner. Nano-Secondaries are conjugated to Alexa Fluor® dyes. Compared to traditional secondary antibodies, Nano-Secondaries facilitate better multiplexing and faster Western blotting.

What is the specificity or target of Nano-Secondaries?

Nano-Secondaries are subclass-specific and do not cross-react with IgGs from other commonly used species. We select only Nanobodies with the desired specificity. Therefore, our Nano-Secondaries have a very low background and do not require any kind of pre-adsorption.

What is one-step immunostaining?

One-step Western blotting is the simultaneous incubation of primary antibody and Nano-Secondary. This method reduces incubation time, hands-on time and the number of washing steps.

Because the Nano-Secondaries are monovalent and bind with high specificity and affinity to their target IgGs, they can be simultaneously incubated with the primary antibody. This results in a one-step Western blotting. In practical terms, the Nano-Secondary is pre-mixed with the primary antibody and the mixture is used for WB staining following the primary antibody’s protocol for Western blotting.

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Spot-tag detection

Spot-Tag is a universal peptide-tag for multiple capture and detection applications. Spot-tagged proteins can be directly detected in Western blotting with Spot Label, ChromoTek’s fluorescently labelled anti-Spot-Tag Nanobody. Alternatively, the unlabeled anti-Spot Nanobody can be used in Western blotting as a primary antibody and can be detected using an HRP-labeled anti-llama secondary antibody.

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Looking for references? Please check our literature database.

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