Analysis of common problems in protein extraction
Q : How to perform tissue liquid nitrogen grinding?
A: Freeze the tissue block in liquid nitrogen, knock it into small pieces of appropriate size, place it in a mortar, pour liquid nitrogen, add it while grinding, keep the liquid nitrogen in the mortar, or keep the tissue block unmelted. . It’s fine to break it up. Be careful to pre-cool the equipment such as the pre-mortem, use liquid nitrogen or put the refrigerator for 20 minutes.
Matters needing attention: 1. The research of liquid nitrogen grinding, the medicine spoon must be pre-cooled. 2. Before starting the grinding, place the mortar on an ice pack and add a small amount of liquid nitrogen to quickly transfer the sample into it. 3. Continue to add liquid nitrogen, quickly mash the sample, and then quickly grind. 4. Before the liquid nitrogen is volatilized, continue to add liquid nitrogen, quickly submerge, and repeat the operation until the sample becomes very fine white dry powder. 5. Scrape the dry powder into the lysate, add the lysate to the mortar, and wash the adhered sample. The whole process keeps the sample in a low temperature environment, and the grinding speed is fast and strong. This will break the cells without degrading the protein.
Q : What if the membrane protein is insoluble in the loading buffer or forms a precipitate after boiling?
A : Denature 1h with 60 degrees ; if it still doesn't work, replace the SDS of the loading buffer with LDS .
Q : How many cell extracts are enough for western blot ?
A: Generally 5 × 10 6 is enough. But depending on which cell, some cells are small, while others are very well spread. It is routine to do a WB with a channel of 10 5 cells. But it is also related to the protein of interest and the way it is extracted. If it is a membrane protein or the number of protein cells distributed in an organelle is increased.
Q : Can the same protein sample be tested by Western Blot of both factors simultaneously ?
A: Yes, you can even measure more than ten samples at the same time.
Q : How long can the protein be stored after extraction?
A: -80 ° C, no problem for one year. Two key points: don't be hydrolyzed by proteases; don't be contaminated by bacteria
Q : What is the total amount of protein that WB is normally loaded after extracting protein , which is related to the expression level of the target protein?
A: Western Blot generally loads between 30 and 100 micrograms. The results are related to the abundance of the target protein, the amount of sample loaded, the amount of secondary and secondary antibodies, and the time of incubation. It is also related to the length of coloration. When you start to touch the condition, in order to get a positive result, each step can be a little longer and longer, of course, the background will come out. To get good results, if the antibodies are good, it is easier. If the antibodies are not good, you need to try them repeatedly. Of course, some of them are not suitable for Western Blot. So getting good results is not easy.
Q : When doing the western phase of the tissue sample protein , is there any special attention to extracting the protein?
A: It must be ground and homogenized, the protein solubility will be better, the centrifugation should be sufficient, and the other is that the protease activity in the tissue is stronger, and it is necessary to pay attention to inhibiting the protease activity (adding PMSF and protease inhibitor cocktail).
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