Comparison of Western Blot automatic analyzer and traditional WB method

Compared with the traditional WB method, the traditional Western blot method uses the antigen-antibody immune reaction, first separates the protein by SDS-PAGE electrophoresis, and then uses the electric field force to transfer the protein on the gel. The solid phase carrier (nitrocellulose membrane, ie, NC membrane) is added to the antibody to form an antigen-antibody complex, and the result is displayed on the membrane or the film by the principle of luminescence or color development. The experimental steps mainly include the following:

one. Rubber running rubber

1. Prepare SDS-polyacrylamide gel according to requirements (usually 10% SDS-PAGE)
2. Load the prepared gel into the electrophoresis apparatus (be careful not to leak).
3. Design the sample loading sequence, make the experimental records, and load the samples in the predetermined order. The general lysate is spotted at 10-20 ul/channel, and the recombinant protein is spotted at 1-2 ug/channel.
4. Connect the electrophoresis device to the power supply. Adjust the voltage to 100V for 10-20min. After the bromophenol blue migrates out of the laminated glue position and replace it with 200V, turn off the power after 30-40min.
5. Remove the gel glass plate from the electrophoresis device, rinse it with water, and prepare to transfer the film.
two. Transfer film

1. Place the gel glass plate in a container containing the electrophoresis transfer buffer and soak for a few minutes.
2, put on the gloves, prepare the filter paper and NC film, try to avoid contamination of the filter paper and membrane, soak the filter paper and membrane immersed in the electrophoresis transfer buffer to remove the air bubbles left on the membrane.
3. Open the transfer box and place it in a shallow dish. Thoroughly soak the sponge pad with transfer buffer and place it on the wall of the transfer box. Place a soaked filter paper on the sponge.
4. According to the order of “sponge-filter paper-gel-NC film-filter paper-sponge” (note that there should be no air bubbles and the electrode slots of the device cannot be reversed)
5. Place the ice box in the buffer tank and fill the 4 °C pre-cooled transfer buffer.
6. The whole device was placed in an ice bath and stirred with a magnetic stirrer. The transfer electrode was connected to a constant current of 300 mA for 90 min. After the electroporation was completed, the NC membrane was well-marked and placed in 5% skim milk powder (PBS preparation), and incubated at 37 ° C for 2 hours or 4 ° C overnight.

three. Closure and hybridization

1. Closed:

The membrane was removed from the electrorotation tank, the deionized water was rinsed slightly with PBST or TTBS, and immersed in the blocking solution and shaken slowly for one hour. If necessary, first observe the protein band with Ponceau red staining (2% acetic acid, 0.5% Ponceau aqueous solution), and then elute the Ponceau with deionized water and TTBS, if omitted with protein marker This step.

2. Combine primary antibodies:

Preparation of primary antibody: When using the reverse-sticking method, about 2 ml of primary antibody dilution is required for each 3×9 cm ² membrane.

Reverse paste operation: a closed droplet containing a primary antibody is applied to the plastic film of the shaker, the Western membrane is taken out from the blocking solution, the filter paper is slightly blotted, and the front side is attached to the primary antibody. The bubbles were incubated at room temperature for 1 hour or at 4 ° C overnight. A wet plate is placed over the reaction system to prevent excessive evaporation of the liquid.

3. washing:

After the incubation of the primary antibody, the membrane was rinsed with PBST or TTBS and then immersed three times for 5-10 min each.

4. Combined with secondary antibody:

Select the appropriate secondary antibody according to the primary antibody source, select HRP or AP labeled antibody according to the identification method, dilute according to the corresponding ratio (1:1000~1:10000), and shake at room temperature for one hour.

5. washing:

After the incubation of the secondary antibody, the membrane was rinsed with PBST or TTBS and then immersed three times for 5-10 min each time.

four. Luminescence identification

One of the most commonly used chemiluminescent labeling enzymes is horseradish peroxidase HRP (Horseradish Peroxidase, which belongs to peroxidase POD) and alkaline phosphatase AP (Alkaline Phosphatase). A darkroom, or a professional chemiluminescent gel imager () and analysis software are usually required for analysis.

It's not hard to see the complexity of the steps. Each of these mistakes will cause a total failure, and the experiment will be repeated. For precious samples, care must be taken. In addition, the test procedure is long and the results are poorly reproducible. To achieve good experimental results, the cost will be high. At the same time, the operation of the required experimental instruments such as electrophoresis, transfer, imaging analysis (or darkroom) is also complicated, which increases the difficulty of the experiment. Under the traditional method, if you want to get a better experimental result, you often need the experimenter to have very high experimental skills and skilled experimental instrument operation experience. Therefore, scientists have been looking for an innovative technology to overcome the shortcomings of Western traditional technology and carry forward its advantages. Until the end of 2011, the US ProteinSimple company specializing in protein analysis and research technology has developed a unique and fully automatic system around the world after years of development. The Western Blot analysis system Simon makes the Western experiment simple and efficient.

Fully automated Western Blot analysis system Simon automates all the experimental steps of Western blot, including protein loading and separation, immunoblotting, washing, detection and quantitative analysis of data, which ensures the repeatability of the experimental results.

The operator simply leaves the sample and then taps "Start" to leave. After only about three hours, you can come back and read the data results that have been analyzed. If you are not sure about the analysis time of the entire process, Simon ^TM will be reminded by the sound after the end of the experiment, it is so simple!

It is mainly to complete all the experiments of Western blot in 400nl sample tube, only need 5ul sample amount to save precious samples; the result is highly reproducible, CV≤10%. The automated sampler has multiple parameters that can be set by itself, including separation time, blocking time, incubation time of primary and secondary antibodies, and detection time.

The analysis of the experimental data is automatically processed by the Compass software, and the representation of the sample data is consistent with the traditional results and has better sensitivity and repeatability. Improve the accuracy of the analytical system's quantification.

After introducing the above two methods, we briefly summarize the comparison between the Western Blot automatic analyzer and the traditional WB method:

First, the degree of automation is different : the traditional WB method requires multiple steps of running glue, film transfer, antibody hybridization, and luminescence imaging and analysis. Each step needs to rely on experience and exploration, and manual operation is required between each step until Get the analysis results. The fully automatic WB analyzer only needs to simply put in the sample and the corresponding program settings, and then the system directly draws the results, the whole process is fully automated, and the experiment process is simple and easy to operate.

Second, the experimental repeatability is different : the traditional WB method, the complicated experimental steps, the long experimental period and almost all the manual operation of the experimental method directly determine its low experimental repeatability. Almost every step of the mistake will result in different experimental results for the experimental results, especially for the primary experimental operators, if you want to get more reliable experimental results, you will almost receive a lot of skill training and many failure lessons.

Third, the experimental time (efficiency) is different : the traditional WB method takes almost two days, and the fully automatic Western blot analyzer only takes a few hours to obtain experimental data.

Fourth, the experimental cost is different : the traditional WB method requires a large amount of sample. In the almost perfect and error-free experimental process, a variety of relatively expensive experimental reagent consumables and a large amount of time and manpower are required. If the experiment fails, the experimental cost is sometimes almost not predictable. The fully automated Western blot analyzer requires no more than 5 ul of sample volume and a small amount of economical experimental reagent consumables. Reliable experimental data can be obtained in a few hours. The experimental cost is low and can be estimated. For some key experiments, almost The maximum guarantee of the experimental program and even the smooth progress of the project.

Of course, the Western Blot automatic analyzer is slightly higher in price due to its professional and strong instrument. At present, its audience is only users with a large number of professional, high-efficiency experimental needs. The various instruments in the ordinary WB method can be used for almost all other gel imaging experiments, and the applicability is wider.