1. Explain what is meant by the terms yield and purity. How are these used in planning a protein purification strategy?
2. If the ASF is successful, will you find the majority of the GFP in a supernatant or a precipitate sample?
3. Precipitate 3 has twice as much total protein as precipitate 4. If the two samples have equal GFP fluorescence, which would you select for further purification and why?
4. The cell lysate sample contains all of the protein originally in the sample, including all of the GFP. Can you have a GFP fluorescence reading in a precipitate sample that is higher than the fluorescence reading from the cell lysate? If so, how is that possible?
Table 1: GFP fluorescence for 100 µl samples from Exp. 3. For use with Questions 9.
|
Sample
|
Dilution
|
Fluorescence, RFU
|
Original sample [GFP], mg/ml
|
|
CL
|
1:10
|
309,246
|
|
|
ASF
|
1:8
|
468,167
|
|
|
ASF-GFC
|
1:2
|
540,256
|
|
|
NACC
|
1:3
|
397,256
|
|
5. Using your RFU vs [GFP] calibration from Experiment 3.1, fill in the last column of Table 1. Which purified sample has the highest concentration of GFP? Which has the lowest concentration of GFP? Include your calibration curve.
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Table 2: Dilutions for loading equal masses of GFP on the SDS polyacrylamide gel. For use with Question 6.
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|
Sample
|
(A) Theoretical RFU of 100µl of the undiluted sample
|
(B) Dilution to get (A) to 500,000 RFU for 100µl
|
(C) volume of sample
|
(D) volume of buffer to add
|
|
CL
|
|
|
|
|
|
ASF
|
|
|
|
|
|
ASF-GFC
|
|
|
|
|
|
NACC
|
|
|
|
|
6. You are preparing samples to load into SDS-PAGE and need to make 100 µl of a solution from each sample that would give an RFU of 500,000 if added to a 96 well plate. Using the date from Table 1 and your GFP calibration data, calculate (A) the theoretical RFU for 100 µl of the undiluted sample; (B) the dilution required to get the undiluted sample to an RFU of 500,000; (C) the volume of sample you will use, and (D) the volume of buffer that will be added to make 100 µl. Show your calculations for completing Table 2.