Protein Expression and Purification Series - Bio-Rad

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CHAPTER 9 DHFR ENZYMATIC ASSAY Protein Expression and Purification Series Absorbance at 340 nm c. Convert this slope from change in absorbance at 340 nm/second to change in absorbance at 340 nm/minute by multiplying by 60. Slope of Control Data x 60 = _____________ Change in Absorbance at 340 nm/minute This will now be referred to as ΔOD, control. d. Look at your data from the “GST-DHFR-His Enzyme Reaction.” Plot the data with the x-axis as Time in seconds and the y-axis as Absorbance at 340 nm. Example data is pictured in Figure 9.4. Note: Ideally, the enzyme reaction data should be linear over the entire range of 150 seconds. As the reaction is set up, it is limited by the amount of DHF. Therefore, if there is a lot more enzyme than substrate, the substrate will be used up before the 150 seconds is over, and the absorbance will not change appreciably for the remainder of the time course (Figure 9.5). If this is the case, calculate the rate only for data before it flatlines (at 95 seconds in figure 9.5 below). If there are enough reagents available, you can also try diluting your GST-DHFR-His sample in 1x PBS and rerunning the entire enzyme reaction. 0.680 0.670 0.660 0.650 0.640 0.630 0.620 Absorbance at 340 nm 0.610 GST-DHFR-His G S T -D H F R -HEnzyme is E n z yReaction m e R e a c tio n 0.600 0 20 40 60 80 100 120 140 160 Time T ime in in seconds se conds Figure 9.4. GST-DHFR-His enzyme reaction. When substrate, enzyme and cofactor are present, the absorbance of the NADPH at 340 nm should decrease if the enzyme is active since the NADPH is being converted to NADP+. From a regression analysis of the above data, the change in absorbance at 340 nm/minute is -4.5x10 -4 /second or -0.027/min. Absorbance at 340 nm 0.540 0.530 0.520 0.510 0.500 0.490 Absorbance at 340 nm 0.480 GST-DHFR-His G S T -D H F R -HEnzyme is E n zReaction y m e R e a c tio n 0.470 0 20 40 60 80 100 120 140 160 Time T ime in in seconds se condsq Figure 9.5. A reaction with too much GST-DHFR-His enzyme. This reaction flatlines, or runs out of DHF, by 95 seconds. After that, the absorbance does not change. The timeframe after 95 seconds is analogous to the No DHF Substrate Control reaction since in that reaction, no DHF was added; while in the above reaction, the GST-DHFR-His converted all the DHF present to THF so there was no more need to convert the NADPH to NADP + and the absorbance values level off. To calculate the rate of the enzyme reaction for this data, the slope of only the data from 0-75 seconds should be used or the GST-DHFR-His should be diluted and the reaction run again if there are enough reagents available. 206 Chapter 9: DHFR Enzymatic Activity Assay Student Protocol
Protein Expression and Purification Series e. Calculate the slope of the line that best fits the GST-DHFR-His Enzyme Reaction data. This may be done by drawing a line that best fits all of the data points being used and determining the change in absorbance at 340 nm over this timeframe or by using a regression computer program or calculator function and determining the slope of the line generated by the data. Record the slope of the Enzyme reaction below. Slope of Enzyme reaction data: _________________Change in Absorbance at 340 nm/second f. Convert this slope from change in absorbance at 340 nm/second to change in absorbance at 340 nm/minute by multiplying by 60. Slope of Enzyme reaction data x 60 = _____________ Change in Absorbance at 340 nm/minute This will now be referred to as ΔOD, reaction. g. Determine the decrease in absorbance at 340 nm due to enzyme reaction. The amount of NADP + produced due to the DHFR in the GST-DHFR-His converting the NADPH to NADP + so that DHF can be converted to THF can be calculated by subtracting the absolute value of the change in absorbance with no substrate present to the absolute value of the change in absorbance with substrate present. Change in absorbance at 340 nm due to enzyme reaction = ΔOD ΔOD = |ΔOD, reaction| - |ΔOD, control| Example: From the data shown in the graphs above, ΔOD, control = 0.002/min so |ΔOD, control| also = 0.002/min ΔOD, reaction = -0.027/min so |ΔOD, reaction| = 0.027/min Therefore ΔOD = |ΔOD, reaction| - |ΔOD, control| = 0.027/min – 0.002/min = 0.025/min. Calculate the ΔOD and record it below: ΔOD (min -1 ) = ________________________ h. Calculate the activity of the purified GST-DHFR-His. The activity of the GST-DHFR-His will be calculated in terms of how many µmol/min of NADPH it can convert to NADP + per ml of reaction volume. This can be determined from a form of Beer’s Law that relates absorbance values to concentration values. In this case, the change in absorbance can be related to the change in concentration of NADPH. Beer’s Law Absorbance (A) = ε x C x l, or for this case ΔOD = ε x (change in C) x l where ΔOD = |ΔOD, reaction| - |ΔOD, control|, which you calculated above Change in C = ΔC = The change in concentration of NADPH over the reaction time course ε (extinction coefficient) = 6220 M-1 cm-1 for NADPH l (length) is the pathlength of the cuvette (usually 1 cm for most cuvettes) Chapter 9: DHFR Enzymatic Activity Assay Student Protocol 207 CHAPTER 9 DHFR ENZYMATIC ASSAY
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Protein Expression and Purification
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CHAPTER 1 BACKGROUND Protein Expres
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CHAPTER 2 FOCUS QUESTIONS Protein E
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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CHAPTER 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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Page 156 and 157: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 210 and 211: APPENDIX A TIPS AND HELPFUL HINTS P
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Page 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
Page 216 and 217: APPENDIX B RESULTS ANALYSIS Protein
Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
Page 226 and 227: APPENDIX F BIOLOGIC LP SYSTEM SETUP
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
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Page 234 and 235: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 242 and 243: APPENDIX I GLOSSARY Protein Express
Page 244 and 245: APPENDIX I GLOSSARY Protein Express
Page 246 and 247: APPENDIX J REFERENCES, LEGAL NOTES
Page 250: Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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