western blot实验步骤（western blot实验步骤）.docVIP

western blot实验步骤（western blot实验步骤） Western blot experimental steps (1) the glass plate with double distilled water scrub, rinse and dry. Mix with 10% ammonium sulfate (preferably with first serving). Install the glass plate on the gel rack, with the inward direction of the groove. Add a small amount of deionized water to see if the water is leaking. If the leak, then reinstall; do not leak, then start sizing. (2) with glue, glue and water cover. With 12% separation gel: gel storage liquid separating gel buffer 2 ml, 1.25 ml, double distilled water 1.75 ml, 10%APS 25 L, TEMED 2.5 L. Shake the solution gently to make it smooth (excess bubbles affect the polymerization). Glue with 1000 l gun. If the separation glue surface is uneven, shake the gel rack by hand and shake the rubber surface [19]. Then with 200 L gun in deionized water (above the gum cover along the glass plate walking sideways, avoid the water surface), can be added to the full of water, so it can be small bubble gum surface. After the separation, the gel is solidified (about 15 minutes will re - reproduce the refractive surface, and then 5 minutes, such as glue fully solidified), pour water and filter paper blot. (3) with 5% concentration gel: gel storage liquid 0.575 ml concentrated gel buffer 0.1675 ml, double distilled water 0.25 ml, 10%APS 7.5 L, TEMED 1.25 L. Concentrate the gel with a 1000 l gun and add it slowly to the separation gel. Then insert the comb to avoid the bubbles. (comb the comb and tilt the comb slightly). After the concentrate is coagulated, the sample is boiled in a boiling water bath for 5-7 min with an added sample buffer and cooled on the ice for at least 5 min. After 10000 g centrifugation, 10 min removed insoluble impurities. The glass plate is transferred to the electrophoresis bath, and a little buffer is used to check for leaks. If leak, remove, reinstall, do not leak, slowly add electrophoresis buffer to full tank. Pull out the comb vertically. The sample is then added on