User:Brigette D. Black/Notebook/Brigettes Notebook/2009/10/09/Bradford Assay Prep

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While the kinesin from Haiqing is great, there is an issue in that we do not have a clear idea of the concentration. She did tell us that the concentration of the total kinesin was about 5M (about .273 mg/ml assuming that the molecular weight of kinesin is about 55 kDa). In order to determine the concentration, I ordered a colometric protein assay from Bio-Rad (# 500-0002), based on the Bradford assay. This kit contains 450 ml of concentrated dye reagent and 28.6 mg of bovine serum albumin.

I reconstituted the BSA standard (#500-0007) to 1.43 mg/ml by adding 20 mL of DI H20 to the poweder, mixing, and lightly vortexing. The manual notes that if the BSA standard is not going to be entirely used within 2 weeks of reconstituting, it should aliquoted and stored at -20C. In accordance with the instructions, I made 19 1 mL aliquots (at 1.43 mg/ml) and stored them in the -20 freezer in a box labeled "BSA Standard".

The dye reagent needs to be diluted one part dye to four parts DI H20. Once diluted, it is good for about 2 weeks at room temperature, the remainder of the dye should be stored at 4 C. I measured out 2 mL of the concentrated reagent and added 8 mL of DI H20 into a falcon tube. I mixed by inverting the tube several times. It is also recommended that the solution then be filtered to remove an extra particulates. I filtered the solution through a 0.2 um filter. We now have about 10 mL of reagent good for about two weeks. The concentrated dye is in the VWR refrigerator.

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