Western, also known as Western blot, Western blotting, Western blotting, is one of the important methods for detecting proteins with antibodies. Western can refer to the following steps.
1. Collect protein sample (Protein sample preparation)
Adherent cells, suspension cells or tissue samples can be lysed using appropriate lysates such as Western and IP cell lysates, RIPA lysates, and the like. For certain specific subcellular component proteins, such as nuclear proteins, cytoplasmic proteins, mitochondrial proteins, etc., these subcellular component proteins can be extracted by reference to relevant literature, or can be extracted using nuclear protein and cytoplasmic protein extraction kits. mention.
2. Electrophoresis (Electrophoresis)
(1) SDS-PAGE gel preparation
SDS-PAGE gels can be formulated using a gel formulation kit that provides all reagents except water and gum dispensers and formulations for various concentrations of SDS-PAGE.
(2) Sample processing
An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples. For example, 2X or 5X SDS-PAGE protein loading buffer. The 5X SDS-PAGE protein loading buffer can be used to reduce the loading volume, and more protein samples can be loaded in the same volume of the well.
Heat at 100 ° C or a boiling water bath for 3-5 minutes to fully denature the protein.
(3) Loading and electrophoresis
After cooling to room temperature, the protein sample can be directly loaded into the SDS-PAGE gel well. In order to facilitate the observation of the electrophoresis effect and the effect of the transfer film, and to judge the molecular weight of the protein, it is preferable to use the pre-dyed protein molecular weight standard.
3. Transfer film (Transfer)
PVDF membrane was used in the Western experiment. A nitrocellulose membrane (NC membrane) can also be used, but the nitrocellulose membrane is relatively brittle, and is easily cleaved during handling, particularly in the process of gripping with tweezers.
The effect of the transfer film can be observed by using the pre-stained protein molecular weight standard. Usually, the 1-2 bands with the largest molecular weight are more difficult to transfer to the film. The effect of the transfer film can also be dyed with the Ponceau red staining solution to observe the actual film transfer effect. The SDS-PAGE gel that has been transferred can also be stained with Coomassie Brilliant Blue Rapid Stain to observe protein residues.
4. Blocking
Immediately after the transfer, the protein film was placed in a pre-prepared Western wash solution and rinsed for 1-2 minutes to wash away the transfer solution on the film. From all steps after the film is finished, be sure to pay attention to the moisturizing of the film to avoid drying of the film, otherwise it will easily produce a high background.
5. Primary antibody incubation
Refer to the instructions for primary antibodies and dilute the primary antibody in a suitable ratio with a Western primary antibody dilution. Exhaust the blocking solution with a micro-desktop vacuum pump or a dropper, immediately add the diluted primary antibody, and incubate for one hour at room temperature or 4 °C on a side-waist shaker with gentle shaking. If the primary antibody is not incubated for one hour, it can be incubated overnight at 4 °C with gentle shaking. The appropriate incubation temperature and time are selected or more according to the instructions of the antibody.
6. Secondary antibody inucubation
The horseradish peroxidase (HRP)-labeled secondary antibody was diluted with a Western secondary antibody dilution in an appropriate ratio with reference to the instructions for the secondary antibody. The secondary antibody needs to be selected according to the primary antibody. For example, if the primary antibody is mouse-derived IgG, the secondary antibody requires selection of a secondary antibody against mouse IgG, such as horseradish peroxidase-labeled goat anti-mouse IgG (HL). Drain the washing solution with a micro-desktop vacuum pump or a dropper, immediately add the diluted secondary antibody, and incubate for one hour at room temperature or at 4 °C on a side-waist shaker.
7. Detection of proteins
Refer to the relevant instructions for the detection of proteins using ECL-like reagents such as BeyoECL Plus. The tableting can be carried out using a dedicated tablet cassette.
8. Membrane recovery
If the protein sample is invaluable, the protein membrane can be treated with a Western primary antibody secondary antibody removal solution to re-use the protein membrane.
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