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Once again, Jose and I are using the protocol generated in December to quantify the protein concentrations my samples and Laura’s samples. We’ll also quantify the protein in Rhonda’s larval geoduck samples. It’s a lot to keep track of! The end goal is to obtain the µL of protein required for 100 µg of protein in our samples, and the corresponding amount of 50 mM NH4HCO3 in 6M urea.
Step 1: Prepare reagents for 90-100 samples
- 1 set of 8 standard vials (recipe in lab protocol)
- 6 mL made (recipe in Laura’s notebook)
- 10 mL made (recipe in Laura’s notebook)
- Need 22 µL 50 mM NH4HCO3 per sample, so 2200 µL or 2.2 mL total
Step 2: Plan microplate arrangement
Figure 1. Microplate arrangement
Step 3: Pipet 10 µL of either standard or sample into the corresponding microplate well
Step 4: Prepare BCA working reagent
The BCA working reagent should only be prepared right before plate incubation and reading.
- ((8 standards x 5 plates) + 100 samples) x (3 replicates each) x (200 µl of working reagent per well) = 104,000 µL working reagent = 104 mL working reagent
- Used 110 mL Reagent A and 220 µL Reagent B to make a 50:1 Reagent A to B ratio.
Step 5: Added BCA working reagent to each well
Figure 2. Microplate 1
Figure 3. Microplate 2
Figure 4. Microplate 3
Figure 5. Microplate 4
Figure 6. Microplate 5
Step 6: Used Genome Sciences incubator and plate reader
Step 7: Calculated volume of protein needed for 100 µg per sample
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Roberts Lab 