Difference between revisions of "Pierce BCA Protein Concentration"

Latest revision as of 16:10, 31 July 2018

Summary

We use this method measure the amount of protein we have in our unknown samples

Method

1. *Place samples in order on ice*
2. Use the sonicator to break up all the stuff in the cell, free the proteins
3. Turn on the machine
4. Clean tip of probe with milli Q water (DI works too), wipe dry
5. Set amplitude at 35% intensity
6. Put on ear muffs. Place tube in probe, hit red button for 10 seconds (hold the tube in place)
7. Place tube on ice
8. Wipe probe. Use water to clean between treatments/ controls. Wipe dry
9. Clean probe and turn off

Method for protein assay

1. Use pierce 660 nm protein assay reagent (brown bottle on bench) and BSA pre-diluted standards (known concentrations. Located in the -20 fridge door, in a box)
2. Use the lysis buffer from the freezer (same as cell culture) as a blank and obtain a 96 well plate (flat bottom)
3. **do duplicated of each solution, loading each solution twice**
4. Load 10 ul of each solution/ sample
5. Start with the blank on the very left side (A1 and B1) ensure there are no bubbles formed
6. Follow with BSA using the lowest concentration first (A2 and B2)
7. Continue with each concentration until done (A3 and B3)...
8. Add proteins the same pattern
9. Place 150 ml of the reagent into an empty box or box lid (make sure its clean)
10. Using a multichannel pipette, quickly add the reagent to each well. The reaction will start right away. Flush each well 10 times to mix
11. Give the reaction about 2 minutes before taking it to the spectrophotometer
12. At the spectrophotometer
13. Place plate in with no cover
14. Open the program
15. Hit file_new
16. Select ‘plate’
17. enter 660 (wavelength)
18. hit mix and enter the plate
19. Select read normal
20. *Once data pops up record all the numbers in your lab book, and trash plate*
21. Use an excel data sheet which calculates concentrations