Publish Time: 2024-05-15 Origin: Site
Only those who have done WB will know how easy it is for WB to overturn! ! ! 'The longest road in scientific research career is the detour of WB experiment...' I want to share some of my experiences with everyone.
#01-Sample Preparation
1. To ensure that the sample protein is protected from degradation, the sample was quickly frozen in a liquid nitrogen environment immediately after collection, and then stored in a -80°C constant temperature freezer. During this process, be sure to configure the positive control sample corresponding to the batch
2. Grind the tissue: Grind thoroughly, add lysis solution (1:10) according to weight, ultrasonic for 1 minute, and place on ice for 30 minutes.
After lysis: ① Observe whether the lysis solution is viscous. The reason for the viscosity may be that there is no ultrasound, or the amount of lysis solution is not enough, and additional lysis solution needs to be added;
②After centrifugation, observe whether the supernatant is clear and whether there is white precipitate below. If it is large, it is not well cracked. After weighing the softer tissue, add the lysis solution in proportion and grind it with a handheld grinder, while the tougher tissue is ground with liquid nitrogen at low temperature.
3. When performing protein quantification, it is recommended to use the BCA (bicinchoninic acid assay) method. Before performing BCA determination, the protein stock solution should be thoroughly vortexed to ensure sample homogeneity. When sampling, the liquid in the middle of the tube should be selected for testing to represent the overall protein concentration. In addition, in order to improve the accuracy and reliability of the measurement, it is necessary to set up duplicate holes (multiple holes) for parallel measurements.
4. Configure the system: Configure the system according to the amount of protein stock solution. Before configuring, be sure to check the group and volume. Concentrate your efforts to prevent adding errors. After configuration, vortex and cook the sample. Remember to add the sample under the liquid surface and do not drop it on the lid.
5. When the prepared samples are not used temporarily, immediately store them in a -20°C refrigerator.
#02-Electrophoresis
1. Mix the electrophoresis solution before use. If the liquid level is too short for the glass plate, it is recommended to use new solution to prevent tank leakage.
2. When making gel in advance, the glass plate must be washed, otherwise it will affect your electrophoresis. When preparing the gel, be sure to mix thoroughly (you can sonicate it appropriately, but not for too long). After adding the separation gel, you can add purified water to flatten the separation gel, so that the separation gel will gel into a straight line (I think this is very important). If the gel is not gelled well, the strips that will run out later will be ugly.
3. Generally, the bromophenol blue will be pressed into a straight line after running for about 20 minutes. You must observe it in the first 20 minutes. If you find that the bromophenol blue is running crookedly, stop in time to check whether the inner tank is leaking and whether the lid is properly closed. You can still save it in the first 20 minutes.
4. Observe whether there is any heating during electrophoresis.
5. Vortex the sample the same number of times before loading. Remember to set a positive control of the same batch.
6. Load the sample slowly, and try to make the sample in each hole rise horizontally. Do not forcefully add the sample at the left and right corners of the hole, otherwise dumbbell bands will appear due to uneven gel holes.
7. After taking the cooked sample out of -20°C, cook it at the deformation temperature for 3 minutes before loading to make it more stable.
#03-Electronic transfer
1. The sandwich structure is clamped, and the filter paper should be replaced in time after fluffing. After development, the two sides are heavy and the middle is shallow. This may be caused by the film transfer clamp being too tight or uneven.
2. Replace the electroporation fluid with a new one if it becomes turbid.
3. Be careful when installing the clamp, otherwise the membrane may shift, resulting in protein loss.
4. During electrophoresis, an ice-water bath is required, and an ice box or ice cubes should be added to the box (ice cubes are recommended) to prevent overheating. Overheating will cause damage.
5. Egg whites.
#04-Sealing and Incubation
1. The appropriate blocking time is about 2 hours. If the time is too short, there will be many non-specific binding sites and the background will be dirty; if the time is too long, the signal will be low.
2. When incubating phosphorylated proteins, it is recommended to use 2% BSA. Skim milk may cause signal loss.
3. The primary antibody incubation time is 16-24 hours. If it is too short, the antigen-antibody binding may be insufficient and the signal may be lost.
4. Wash the membrane several times in a short time, and change TBST frequently. If the elution effect is not good, you can add a little more Tween20.
5. Do not rush all the liquid directly onto the membrane, add it from the edge of the container, or slowly add it from the edge of the membrane.
#05-Exposure
2. If the band signal is low, use a more sensitive luminescent solution and extend the exposure. Consider: excessive blocking, increasing primary antibody concentration, incubation time, and increasing secondary antibody concentration.
3. If there are black spots and white spots, it is caused by uneven luminous fluid.