Reference no: EM1394362

You measured the purified protein concentration, which is 5 ug/uL. You decided to load 20 ug protein to the gel. How would you dilute your sample? Keep in mind that you also need to add the 2 x sample buffer to the protein, and the total volume to load in the gel is 20 uL. (2 pts)
normal procedure is:
Experimental Procedures:

Reagents:

1. Tris Buffer pH 8.2 - located on TA's bench
2. Your BEW, Pool A and pool B samples from EXP5 - located on TA's bench
3. 2X Sample denaturing/loading buffer - located at the center of your bench
4. Reservoir buffer (will be used by TAs)
5. Protein size standard markers mix (ready for loading to the gel) (will be used by TAs)
6. Coomassie Blue Stain solution (0.1% Coomassie blue, 42% methanol, 15% acetic acid) (will be used by TAs)
7. Destaining solution (30% methanol, 10% acetic acid) (will be used by TAs)
Equipment:
1. Electrophoresis Box - located on your bench
2. Power supply - located on your bench
3. Micropipettes 20 - 200ml and 200-1000ml - located in your shared equipment drawers
Glassware and Plastic
1. 1.5 ml microcentrifuge tubes - located in the rack at the center of your bench
2. Racks for 1.5 ml microcentrifuge tubes - located in you drawer
Sample Preparation: You need to prepare 1 ml of 1 mg/ml of: BEW, Pool A and Pool B from EXP5.
You prepared protein dilution table in your EXP5 Lab Report. Please, show this table and corresponding calculations to instructor or TAs. We want to be sure that protein concentration is properly calculated.
Label 40 ml beaker as Tris Buffer and transfer about 10 ml of Tris Buffer pH 8.2 to this beaker. (We recommend using paper tape to label any glassware during this course).
1. Label three 1.5 ml microcentrifuge tubes as BEW, Pool A and Pool B.
2. Prepare BEW, Pool A and Pool B dilutions according to your protein dilution table. Use TRIS Buffer pH 8.2 for dilution. Mix samples using vortex.
3. Label 3 additional 1.5 microcentrifuge tubes as BEW, Pool A and Pool B.
4. Add 50 µl of diluted BEW, Pool A and Pool B from step 2 to the corresponding tubes from step 3.
5. Add 50 µl of 2X sample denaturing buffer (SDB) to each of tubes from step 4, mix solution by pipetting.
6. Heat samples from step 5 for 7 min at 95°C using dry block heater.
7. You will load 20 µl (using 2-20ml micropipette) of each prepared in step 6 samples into the Gel wells designated for your group. Protein size marker will be loaded by TA (10 µl/well).