Thank you very much, Sebastian Schmitt

From my experience if you have to perform one, two western every week I suggest you to prepare at least 500ml of 10x running and trasfer buffer, but if you have to perform few westerns, or one every months. It is better to prepare less solution (100-200ml). When the running and transfer recipe turn yellow it is due to the glycine and the pH changing and you have to discard all the solutions. You risk to waste reagents.

For Laemmli buffer I prepare usually the 5x without Mercaptoethanol and store at RT or at -20°C and I add Mercaptoethanol when preparing the samples. You can store it longer wihout Mercaptoethanol.

A suggestion for cold 1X transfer buffer, it can happen, that with all the thing to remember, you forget to prepare cold transfer buffer and you don't have time to cool it. Leave always a 1L bottle of distilled water in the fridge, you can prepare 1x transfer buffer and put in the fridge also the last minute.

For 10x TBS-T or PBS-T I prepare the solution without tween and add it in the 1x, it is better

João Pedro Simões

Similar to what Sebastian mentioned, with Western blot reagents you want to make sure that you avoid contamination of solutions, to avoid contamination you have to prepare the buffers in a volume that is reasonable.

Lysis buffer can be a few weeks to months depending on what type of buffer you are using and as long as you are only adding any protease inhibitors or PMSF fresh

SDS running buffer you can prepare a 10x stock solution and keep at room temperature and it is shelf stable for months as long as you dont see the SDS precipitate out. If you see crystals at the bottom, remake it and store in a location within the lab that has temperature stability.

Transfer buffer contains methanol and like the running buffer, you can create a 10X solution without methanol and store it at room temp for weeks to months, and just add the methanol fresh. Otherwise you can make a 1X Transfer buffer and store it at 4 degrees for a week or two

For blocking buffer (Usually 5% BSA or Nonfat dried milk) can again be made as a 10X stock solution of your PBS-T or TBS-T and add the BSA or milk fresh. Once you add the milk, you can store at 4 degree for a day or so (but just like milk sitting in a fridge at home, it has a limited shelf life).

For your wash buffer/antibody dilution buffers you can create a 10X TBS and TBS-T solutions that are shelf stable for a long time.

Basically if you keep the lids on tight with your buffer stocks (10X solutions or Lysis Buffers) you can have a long shelf life. But if you start experiencing unexpected results make everything fresh. Once you start running western blots more, you will find out how fast you are going through certain buffers and can adjust volumes accordingly. Hope this novel of a response helps a little bit

Thsi strongly depends on conc., sterility in you lab ans storage conditions (RT, 4°C etc.). To get optimal results, I would propose to prepare solutions freshly.