Hello,

If I am reading this correctly, you have a 2X STOCK of cOmplete Ultra and 10X STOCK of Phospho-Stop.

So if you require 10 mL of lysis buffer (Including all inhibitors). Then set Vf (volume final) to 10 mL.

For Vf of 10 mL you require:

- 1 mL of 10X STOCK Phospho-Stop (10 mL / 10X stock = 1 mL)

- 5 mL of 2X STOCK of cOmplete Ultra (10 mL / 2X stock = 5 mL).

That leaves 4 mL remaining. This 4 mL remaining will be your lysis buffer solution.

EDIT: But now I wonder if that leaves to little lysis buffer which may be what you are thinking...maybe you could add the 1 tablet of cOmplete Ultra straight into 9 mL of lysis buffer + 1 ml of 10x Phospho-Stop?? Anyone can comment/confirm?

EDIT 2: Or just add a tablet of each into 10 mL of lysis buffer, that could be sufficient?

The amount you need to add depends on the strength of the stock (2x and 10x accordingly). @Aaron Dhanda has made a note of this process.

In these isolated cases where the stock concentration is not strong enough, you can ignore the factory suggestions and make the stock as concentrated as possible. You can add the solvent in small increments and periodically vortex the mixture till the solute is completely dissolved. Keep a note on the volume you are adding so that the strength can be back calculated. Make sure you use these concentrated stocks immediately after making them. It is never advised to store these stocks for longer periods as they will preciptate out your solute with time and lowering temperatures. So, to minimise loss, you can cut these tablets and weigh them in percentages according to your lysis buffer's requirements. Some of them are soluble in your lysis buffer directly as well.

Thank you very much Aaron and Praveesh for your valuable answers. I recently contacted the manufacture asking about more apparent details with regard to my points and got that from them.

I'll post it here for anyone who might need that information:

For phosstop, in the user guide you can see : ----------------- Preparation of Working Solutions : Use one tablet to inhibit phosphatase activity in 10 ml extraction solution... Preparation of 10X conc. Stock Solution :  Dissolve one PhosSTOP tablet in 1 ml water or buffer via swirling. ---------------- So, with 1 tablet you prepare 10 ml of lysis buffer. If you prepared the 10X solution, you then use 100 µl of 10X solution per ml of lysis buffer.

For the Complete tablet, this is the same principle. From the user guide :with the Complete tablet : -----------------   Direct Use with Lysates : One cOmplete ULTRA Tablet is sufficient for the inhibition of the proteolytic activity in 50 ml extraction solution. When very high proteolytic activity is present, 2 tablets may be used in 50 ml of extraction solution. Tablets can be added directly to the extraction medium. Preparation of a Stock Solution (2X concentrated) Alternatively to the direct use with lysates, a 2 stock solution can be prepared, with the following procedure: Add one cOmplete ULTRA Tablet to 25 ml double-distilled water in a PE or PP tube. ------------------------- So with one tablet, you prepare 50 ml of lysis buffer. If you prepared the 2X solution, you use 25 ml of 2X stock solution + 25ml of lysis buffer.

With both products, keep in mind that if you choose to prepare the 10 X and 2X solutions, you'll then affect the composition of your lysis buffer (especially with the 2X Complete).