Lab 9: PROCEDURE
Assay Overview:
- Start by planning your experiment:
- You will be using a 96-well plate, and each well will contain a single test condition. Create a detailed chart like the one below to keep track of the volumes of solution you are adding to each well (this should be completed before lab).
| Plate Row | Plate Column | Protein | Protein Stock Conc | Protein Volume | Urea stock Conc | Urea stock Volume | Protein Buffer | Final Urea Conc |
| A-B | 1 | Blank | 0 | 0 | 0 | 0 | 100 μL | 0 |
| C-E | 1 | Wt | 5 μM | 10 μL | 0 | 0 | 90 μL | 0 |
| … | ||||||||
| A-B | 2 | Blank | 0 | 0 | 0.56M | 90 μL | 10 μL | 0.5 M |
| C-E | 2 | Wt | 5 μM | 10 μL | 0.56M | 90 μL | 0 | 0.5 M |
| … |
-
- Organize your experiment to minimize pipetting: add the same concentration of urea in each column and add equal volumes of the same component (i.e. buffer blank or protein) to each row.
- Suggested rows: A and B – Control, C to E – wild type, and F to H – mutant HCAII
- Plan for a final volume of 100 μL per well.
- Choose final protein concentrations. The final concentration of protein in each well should be 0.8 – 2.0 µM. The higher the protein concentration, the better your data will be; however, you still need to have protein for future labs. If you have at least 1 mL protein at a concentration of 50 µM or higher (or the equivalent), use a final protein concentration of 2.0 µM. If you do not have that much protein, use a lower concentration. Talk to the teaching staff if you are not sure what to do. Note that you can use different concentrations of protein for wild type vs mutant, if necessary.
- Once you’ve decided on final protein concentrations, make 500 μL stocks that are 10x concentrated using your protein stock from lab 8. Make sure to keep your proteins on ice and invert the tube a couple times before making the 10x stock. For example, for a final protein concentration of 2.0 µM, make a 20 µM stock. Using a multichannel pipette, add 10 µL of this stock of wild type or mutant, or protein buffer, to the designated rows.
- The remaining volume to 100 μL will be different concentrations of urea solution. You should test 11 concentrations of urea between 0.5M and 9M (plus 0M). Figure out the volume of urea you can add to each well, and then what urea stock concentrations you want to use.
- Test each urea concentration in triplicate, unless you do not think you will have enough protein to complete the rest of the labs. If you are not sure, discuss with your TAs.
- You will be provided with 10 mL of 10M urea in protein buffer. Use this stock urea solution to make several 1 mL dilutions in Protein Buffer, creating the stock solutions you planned in your chart. Again, you should test 11 concentrations between 0.5M and 9M.
- Pipette the appropriate urea solution into each column with a multichannel pipette. Conduct pipetting with increasing urea concentrations and rinse/dry the reservoir prior to adding the next stock. The following video demonstrates how to use multichannel pipettes to set up a 96-well plate:
- Pipette the appropriate protein into each row using a multichannel pipette. Pipette up and down to mix the solutions – being careful not to create bubbles.
- Cover your plate with labeled plastic wrap and incubate at room temperature for 60-90 minutes.
- Use the plate reader to excite at 280nm and measure emission from 310nm-370nm in each well.