User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/23/κ Casein and Buffers
Today I thought it was just about time to break out the κ casein and make some new buffers.
Suspending the κ Casein
A big problem with casein is that nearly all types of casein are insoluble in water, so we have to suspend ours in BRB80. I used the new BRB80 that we made yesterday for everything, so hopefully we did not mess anything up.
I wanted to make a 1 mg/mL solution, so I weighed out 3.3g of the κ casein and then pipetted in 3.3 mL of BRB80. I vortexed the solution until all the chunks were dissolved, and then filtered it through a 0.2 um filter two times (after the first time there still appeared to be some chunks that had not entirely dissolved). After losses by bubbles from vortexing and filtering, I had almost exactly 3mL
- Steve Koch 12:00, 25 June 2009 (EDT): It's worrisome that there chunks visible, although I do recall the bottle saying that the product was only ">80% kappa casein." After we learn how to use the spectrophotometer with the kinesin activity assay, we can purchase reagents for the "Bradford Assay" (or similar), which will allow you to measure the amount of protein (roughly) in the solution.
- BRB80+ 0.5 mg/mL casein
To make this I added another 3 mL of BRB80 to the κ casein solution, for a total of 6 mL BRBCS0.5. I put a total of 2 mL of this into two aliquots (1.5mL and .5mL), and placed these in the refrigerator.
I made up a new buffer that I am simply calling BRB80C. It is made of:
- BRB80 + 0.2 mg/mL κ casein
This seemed like a useful thing to make and store because I can very easily make BRB80CT and BRB80CA from this solution. I used 4mL of the BRB80CS0.5 and 6mL of BRB80. I then made 10 aliquots (1mL each) and put these in the refrigerator. Now, to make BRB80CT all that needs to be done is to add 5uL of taxol to one of these aliquots. Also, to back BRB80CA, one simply needs to add 10 uL of MgATP to one of the aliquots. I figured this would help preserve the buffers since taxol and MgATP can lose their potency when they are not frozen.
- Steve Koch 12:00, 25 June 2009 (EDT): I think this is a great idea!
I took one of the aliquots of BRB80C and added 5uL of taxol. Since I added the taxol to a solution already at 1mL, the concentration is actually 9.95uM, but this seems close enough.
I made 1mL of BRB80T by adding 5uL of taxol to 1mL of BRB80. Again the concentration is slightly less than the recipe says, but only by .005uM, so again this should be close enough.