Laboratory Analysis of Multi-Column Purification of rGFP

BIOL 3380 Experiment 8 - Lab Report Multi-column purification of rGFP Name: Circle your lab section: T-AM W-AM R-AM F-PM T-PM W-PM R-PM F-PM Instructor: Jing Pan Graduate TA: Date: 11/14/23 8-1

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Experiment 8 – Lab Report Multi column Purification of rGFP To save time, simply edit this word document by inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate. 1. (1pt) Attach/inbed an image of your RAW RFU data sheet from Lab 4 (see Lab Report 4 Question 6). 8-3

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2. (1pt) Based upon the protocol, which two fractions should you pool from column 1 (Ni +2 agarose) to apply to column 2 (anion exchanger)? E2 and E3 3. (2pts) Based upon the protocol, what was the total amount of rGFP activity that you loaded onto column 2? Show your calculations for credit. E2 – 70218 RFUs / 200 ul = x / 500 ul = 175545 RFUs E3 – 40267 RFUs / 200 ul = x / 500 ul = 100,668 RFUs Pool – 175545 + 100,668 / 2 = 138,106 RFUs 4. (3pts) Based upon the protocol, if the highest fluorescence was found in fraction F32, does rGFP carry a net positive, net zero, or net negative charge? Explain your reasoning for full credit. rGFP carries a negative charge because sufficient Cl-loading onto the column causes fraction F32, which binds to the positive column without competing with the negative rGFP. 8-4

Use the elution profile above for the Sephadex G-100 Column to answer the following: 5. (3pts) Based upon the protocol, predict a reasonable void volume for the Sephadex G-100 column? Explain your reasoning for full credit. About 25ml, as this is the protein-free portion that comes before peak 1 and does not peak. 6. (3pts) Which peak represents the largest protein/protein complex? Explain your reasoning. Out of the three protein complexes, Peak 1 eluted first, showing that it had bypassed the pores that were more accessible to the smaller proteins. 7. (2pts) What could you do in future purification procedures to sharpen peak 2 like peak 1? Explain your reasoning. Raising the flow rate causes the protein to elute more quickly. As a result, we can observe that a higher flow rate produced a sharper peak (which is peak 1) while a lower flow rate produced a broader peak (which is peak 2). 8-5

Step Sample Total protein (ug) Based on Bradford Assay Activity (RFU) based on fluorescence % purity based on SDS-PAGE A GCE (1ml) 790 4,800 10 B Pooled E fractions from Ni +2 Agarose less 98 3,500 20 C Pooled F fractions from anion exchanger 40 3,000 85 D Pooled GF fractions from Sephadex G- 100 6 2,900 90 Using the Exp 8 protocol and the purification table above to answer the following questions: 8. (2pts) How much rGFP in ug was present at the beginning of the purification process? Show your calculations for credit. 790 ug X 0.05 purity = 39.5 ug rGFP 9. (2pts) Going from what two steps resulted in the least loss of activity? Going from step C to D. 10. (2pts) Based upon the SDS-PAGE, going from what two steps resulted in the largest fold increase in purity? Going from step A to B. 11. (2pts) Going from what two steps resulted in the greatest fold decrease in total protein? Show your work for credit. Going from step A to B. A-B 790 ug/ 98 ug = 8.06 B-C 98 ug/ 40 ug = 2.45 C-D 40 ug/ 5 ug = 8 12. (2pts) What was the yield in ug of rGFP finally purified using this purification process? Show your calculations for credit. Yield of 4.5 ug. 5 ug x 0.9 =4.5 ug 8-6

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13. (2pts) Is specific activity a good indicator of increasing purity? Yes/No. Show your work for credit. Yes. A – 4800 RFUs/ 790 ug = 6.08 RFUs/ug B – 3500 RFUs/ 98 ug = 35.71 RFUs/ug C – 3000 RFUs/ 40 ug = 75 RFUs/ug D – 3900 RFUs/ 5 ug = 580 RFUs/ug 14. (3pts) In an attempt to save money and time, which step would be the best step to remove without significantly altering the purity of rGFP? Justify your answer Step D because based on the SDS-PAGE, there was only a 1.06 fold increase in purity from step C to D and thus, was the lowest fold increase out of the other steps. Step A-B had an 8 fold in purity and step B-C had a 2.125 fold increase. 8-7

BIOL 3380 lab report 8

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