What is the reason for the dirty background in the WB test? How can I fix it?
The Western Blot experiment can be said to be the most basic experiment in the biology major. However, although WB experiments are done every day, a normal high-definition result picture is not something you can have whenever you want.
The most common problem with WB experiments is various background issues.
Just imagine, after finally getting through all the steps and arriving at the final step, the scan result comes out like this:
Why is the background dirty even though the desired trend has been achieved? How can I prevent my strip from being ruined by the dirty background?
It should be noted that a dirty background is not necessarily caused by unclean equipment. It may also be caused by protein degradation or unsuitable conditions during the entire experiment. There may be problems in the following areas:
1. Experiment preparation
The grinding equipment required for protein extraction, as well as the glass plate and comb used to make the gel, should be cleaned thoroughly.
If the equipment is not used for a long time, it is recommended that it be cleaned again before the experiment to prevent corresponding contamination.
2. Protein extraction
Select an appropriate protein lysis solution to fully remove some interfering factors, such as nucleic acids, polysaccharides, lipids, etc.;
It is recommended that the protein be denatured and divided for storage after measuring the concentration to avoid repeated freezing and thawing that causes protein degradation;
Pay attention to the expiration date of the loading buffer. After the protein is added to the loading buffer, mix thoroughly and then boil to denature.
3. Gel preparation
After the glass plate is clamped, some people are in the habit of adding water to see if there are any leaks. It is recommended to use distilled water instead of tap water for testing. After the test is complete, pour the water between the glass plates cleanly and blot the remaining water with filter paper.
Before pouring the glue, mix the prepared reagent thoroughly. During the process of pouring the glue, control the speed and add slowly to avoid creating bubbles.
After pouring the separating gel, slowly add anhydrous ethanol to seal the gel. This step must be done slowly to prevent the gel surface from being deformed by washing; do not keep shaking the gel while waiting for it to set;
At higher temperatures, it will set in about 30 minutes, but in winter, when the temperature is lower, it may take longer;
If the surface of the separating gel is not level, it will affect the results of the later protein electrophoresis.
4. Transferring the membrane
Before transferring the membrane, place 3-4 filter papers on each side of the transfer plate (the same number on both sides);
Remember not to touch the PVDF membrane and filter paper directly with your hands, as the grease on your hands will contaminate them. Be sure to wear clean gloves and use tweezers as much as possible throughout the process;
During the transfer process, the bubbles in each layer of sponge-filter paper-gel-PVDF membrane-filter paper-sponge must be emptied;
The entire electrophoresis instrument must be placed in an ice-water mixture during the transfer process, which is usually carried out using 220V or other high voltage;
An increase in the temperature of the instrument will cause protein degradation. That is to say, after the transfer, if you stain with leishunhong, you will find that the bands are smeared, and after the bands glow, they and the background will also be dirty and messy.
5. Incubate the antibody
Most people choose to incubate the primary antibody at 4°C overnight, but it can also be sealed at room temperature for 2 hours. The effect of incubating at 4°C overnight is better than at room temperature. There is actually no hard and fast rule for how long it is appropriate to incubate at 4°C overnight.
The incubation time for the secondary antibody should not be too long. It is generally incubated at room temperature on a shaker for 1 to 2 hours. After the antibody incubation is complete, it should be washed clean, especially after the secondary antibody incubation is complete, otherwise it may cause a high background in the development results.
6. ECL solution preparation
Most laboratories use ECL luminescent solution, with A and B liquids being mixed in a 1:1 ratio.
The luminescent solution should be prepared just before use, with the amount of solution used being reasonable for each use.
If the luminescent solution is prepared for too long, the luminescence effect will also be poor, with a dirty background or a dark field.
For more product details, please visit Aladdin Scientific website.