1. The effect of blocking with skim milk powder is not necessarily worse than that of BSA, but we have tried several kinds of milk powder, some milk powder is not suitable. Recommended milk powder: Nestle high calcium milk powder, multi-power low-fat milk powder. General domestic The powder of milk powder is relatively large and is not easy to dissolve.
2. About the three doctors: I used to spend a lot of time on preventing short circuits. When I did it, I didn’t cut it at all. The most important thing was to catch bubbles. This is the key. In fact, the principle of the film is current. One-way flow, the electrode of a lot of company's film transfer device is a flat surface, so don't worry about short circuit. (If you don't believe it, try it!!)
3. About the temperature at the time of film transfer: The temperature at the time of film transfer is important, and it is preferable to keep it at 10 degrees or less. You can use an ice bath (Pharmacia's film transfer unit), or you can put in the appropriate preparation in the transfer liquid. Ice pack (-70 degrees > 3 hours) (Bio-Rad), which can both cool down and save Buffer.
4. Regarding the film current and time: the current of the general film is between 200mA and 400mA for 1 hour, the large segment of >50KD can be used for 350mA, and the small segment can be used for 250mA, but I think the above current can be very high. Good to transfer most of the protein.
5. About repeated labeling and re-labeling: A membrane can be labeled with many antibodies at the same time, but it should be noted that generally the difference between two proteins in the target is 5KD or more, preferably two antibodies are different secondary antibodies. When the target fragments differ by less than 5KD, they need to be treated and then labeled (Re-Blot). Some companies have re-labeled ready-made reagents, but they are more expensive. The simplest method is: ddH2O wash for 5 minutes, 0.2M NaOH for 5 minutes, then Use ddH2O5 minutes * 3 times, then you can mark it again. To make a choice of membranes for multiple antibodies, it is best to use PVDF or teach strong, it is recommended to use Amsham's Hybond-P, Hybond-Extra. I am at most one Make 10 different antibodies on the membrane.

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