The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present. The paper will first describe the protocol for western blot, accompanied by pictures to help the reader and theory to rationalize the protocol.
This will be followed by the theoretical explanation of the procedure, and in the later section, troubleshooting tips for common problems. Protein can be extracted from different kind of samples, such as tissue or cells. Below is the protocol to extract proteins from adherent cells. Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline PBS and rocking gently.
Discard PBS. Tip: Keep tissue culture dish on ice throughout. Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes. Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail. Mix well. Add stacking gel solution carefully until the level is equal to the green bar holding the glass plates [ Figure 2 ]. Add H 2 O to the top.
Wait for 15—30 minutes until the gel turning solidified. Tip: Using a suction pipette can make the process of adding the gel to the glass plate easier. Overlay the stacking gel with the separating gel, after removing the water. Tip: It is better to tilt the apparatus and use a paper towel to remove the water. Wait until the gel is solidified. Tip: Solidification can be easily checked by leaving some gel solution in a tube. Pour the running buffer into the electrophorator [ Figure 3 ]. Place gel inside the electrophorator and connect to a power supply.
Tip: When connecting to the power source always connect red to red, and black to black. Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel [ Figure 6 ].
Cut 6 filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride PDVF membrane with the same dimensions. Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol.
Tip: Ensure there are no air bubbles between the gel and PVDF membrane, and squeeze out extra liquid. Add transfer buffer to the apparatus, and ensure that the sandwich is covered with the buffer. Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode [ Figure 7 ]. Transfer for 90 minutes [ Figure 8 ]. Tip: The running time should be proportional to the thickness of the gel, so this may be reduced to 45 minutes for 0.
Wash the membrane with TBST for 5 minutes. Do this 3 times. Tip: All washing and antibody incubation steps should be done on a shaker at room temperature to ensure even agitation. Incubate the membrane for 1—2 minutes [ Figure 10 ]. Visualize the result in the dark room [ Figure 11 ].
Tip: If the background is too strong, reduce exposure time. Cell lysates are the most common form of sample used for western blot.
Protein extraction attempts to collect all the proteins in the cell cytosol. This should be done in a cold temperature with protease inhibitors to prevent denaturing of the proteins. Since tissue sample display a higher degree of structure, mechanical invention, such as homogenization, or sonication is needed to extract the proteins. After extracting the protein, it is very important to have a good idea of the extract's concentration.
This eventually allows the researcher to ensure that the samples are being compared on an equivalent basis. Protein concentration is often measured using a spectrophotometer. Using this concentration allows to measure the mass of the protein that is being loaded into each well by the relationship between concentration, mass, and volume.
After determining the appropriate volume of the sample, it is diluted into a loading buffer, which contains glycerol so that the samples sink easily into the wells of the gel.
A tracking dye bromophenol blue is also present in the buffer allowing the researcher to see how far the separation has progressed.
The sample is heated after being diluted into a loading buffer, in order to denature the higher order structure, while retaining sulfide bridges. Denaturing the high structure ensures that the negative charge of amino acids is not neutralized, enabling the protein to move in an electric field applied during electrotransfer.
It is also very important to have positive and negative controls for the sample. For a positive control a known source of target protein, such as purified protein or a control lysate is used. This helps to confirm the identity of the protein, and the activity of the antibody. Western blot uses two different types of agarose gel: stacking and separating gel. The higher, stacking gel is slightly acidic pH 6. The lower gel, called the separating, or resolving gel, is basic pH 8.
Protein is thus separated by their size more so in this gel, as the smaller proteins to travel more easily, and hence rapidly, than larger proteins.
The proteins when loaded on the gel have a negative charge, as they have been denatured by heating, and will travel toward the positive electrode when a voltage is applied.
Gels are usually made by pouring them between two glass or plastic plates, using the solution described in the protocol section. The samples and a marker are loaded into the wells, and the empty wells are loaded with sample buffer. The gel is then connected to the power supply and allowed to run. The voltage is very important, as a high voltage can overheat and distort the bands.
Typically, this is done using a solution of five percent milk or bovine serum albumin, BSA, for two hours at room temperature or overnight at four degrees. The time and type of blocking buffer should be optimized, so check the datasheet of the primary antibody you intend to use for details. After the membrane is blocked, remove the blocking buffer and add the diluted primary antibody in the same solution.
Incubate on the rocker as before. Typically primary antibody incubations are for one hour at room temperature or overnight at four degrees C. Antibody concentration and incubation time will need to be optimized. Refer to the antibody datasheet for guidance. Pour off the primary antibody and rinse the membrane twice in wash buffer.
Follow with one minute wash and three 10 minute washes on a rocker. Pour off the wash buffer and incubate the membrane in conjugating secondary antibody which has been diluted in blocking buffer.
Usually, this is done for one hour at room temperature, but antibody concentration and incubation time will need to be optimized. Pour off the secondary antibody and wash the membrane as shown previously. There are several different systems for detection.
If the secondary antibodies conjugate into an enzyme, incubate the membrane in the appropriate substrate before imaging. If the secondary antibodies are fluorescent conjugates then you can move directly onto the imaging step. Imaging can be carried out with X-ray film or with a digital imaging system.
Place the membrane into an imaging tray. Place the imaging tray into the imaging system. Exposure times will most likely need to be optimized in order to clearly detect the bands relating to the proteins of interest. Western blot resources.
Buffer and stock solutions. Recommended controls. Western blot sample prep. Transfer and staining. Membrane stripping. Troubleshooting tips. Primary antibodies for western blot. Loading controls for WB. Anti-beta Actin ab loading control. Recombinant antibodies - the benefits.
Secondary antibodies for western blot. Find the right HRP secondary antibodies. Guide for fluorescent WB. Western blot training. Our western blot protocol includes solutions and reagents, procedures, and useful links to guide you through your experiment. Reviewed December 14 Western blotting is a technique that uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis.
Selection is by choosing animal, immunization schedule, affinity purification, performance in a particular assay Western Blot, ELISA, IF, flow cytometry, paraffin section staining , choice of clone if monoclonal, lot if polyclonal. You may have to use an antibody which has not been selected for the use you want. With a monoclonal antibody, affinity constant for the antigen and crossreactivity with unrelated antigens are fixed values, being the immunoglobulin clonal.
If you have a monoclonal with high affinity, as little as 5 minutes may be enough. However, we noticed negative staining with strong antibodies incubated for as much as one hour, compared with overnight incubation e. This may be due to a combination of factors Ag density, masking, affinity. We use almost invariably high dilutions and overnight incubations.
0コメント