HMG-CoA reductase assay

HMG-CoA reductase assay

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(New page: '''HMG-CoA-reductase -assay''' 1. You need a 10 cm dish of cells (60-240µg of protein of total membranes). Remember replicate samples.<br>2. Grow cells ~ 40 h in lipoprotein deficient me...)
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'''HMG-CoA-reductase -assay'''
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'''HMG-CoA-reductase -assay'''  
1. You need a 10 cm dish of cells (60-240µg of protein of total membranes). Remember replicate samples.<br>2. Grow cells ~ 40 h in lipoprotein deficient medium<br>3. Keep the cells on ice. Wash cells twice with cold PBS <br>4. Collect cells by scraping in 5 ml PBS and wash the dish again with 2 ml of PBS. Move cells (and washes) to 15 ml Falcon tubes. <br>5. Pellet cells 2000 x g 5 min + 4ºC. You can freeze the cell pellets in -70ºC.<br>6. Lyse cells in 1 ml of 20mM Tris-HCl + 1 mM EDTA pH 7.5. Enhance the lysis by passing the lysate 20 times through a 21G syringe needle.<br>7. Transfer the lysates to ultracentrifuge tubes. You can increase the volume of the lysates with 1 ml of lysis buffer if necessary.<br>8. Ultracentrifuge 1 h at 100 000 x g in +4ºC to collect total membranes.<br>9. Discard the supernatant and either freeze the pellets in -70ºC or continue with protein assay and do the HMG CoA reductase assay during the same day  
1. You need a 10 cm dish of cells (60-240µg of protein of total membranes). Remember replicate samples.<br>2. Grow cells ~ 40 h in lipoprotein deficient medium<br>3. Keep the cells on ice. Wash cells twice with cold PBS <br>4. Collect cells by scraping in 5 ml PBS and wash the dish again with 2 ml of PBS. Move cells (and washes) to 15 ml Falcon tubes. <br>5. Pellet cells 2000 x g 5 min + 4ºC. You can freeze the cell pellets in -70ºC.<br>6. Lyse cells in 1 ml of 20mM Tris-HCl + 1 mM EDTA pH 7.5. Enhance the lysis by passing the lysate 20 times through a 21G syringe needle.<br>7. Transfer the lysates to ultracentrifuge tubes. You can increase the volume of the lysates with 1 ml of lysis buffer if necessary.<br>8. Ultracentrifuge 1 h at 100 000 x g in +4ºC to collect total membranes.<br>9. Discard the supernatant and either freeze the pellets in -70ºC or continue with protein assay and do the HMG CoA reductase assay during the same day  
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'''Determining the protein concentration'''<br>10. Resuspend the membrane pellets in 150 µl of cold 50 mM K-phosphate + 5 mM DTT (fresh) + 1 mM EDTA (difficult to resuspend)<br>11. Protein assay can be done with BIO-RAD DC protein assay –kit. <br>12. Dilute the samples (just the amount you need for the protein assay) 1:5 in water, because 5mM DTT disturbs the protein assay. <br>13. Dilute also the standards so that you get them in 1/5 x resuspension buffer.<br>14. Vortex the samples well before taking a sample for the assay
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'''Determining the protein concentration'''<br>10. Resuspend the membrane pellets in 150 µl of cold 50 mM K-phosphate + 5 mM DTT (fresh) + 1 mM EDTA (difficult to resuspend)<br>11. Protein assay can be done with BIO-RAD DC protein assay –kit. <br>12. Dilute the samples (just the amount you need for the protein assay) 1:5 in water, because 5mM DTT disturbs the protein assay. <br>13. Dilute also the standards so that you get them in 1/5 x resuspension buffer.<br>14. Vortex the samples well before taking a sample for the assay  
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'''Assay'''
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'''Assay'''  
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15. Use 150 µg of protein/assay. If there is not enough protein in 40µl, 50µg is enough.<br>16. Combine the following reagents on ice in 1.5 ml tubes. Final volume is 102µl. <br>&nbsp; -10 µl 25 mM NADP – 200 mM glucose 6-phosphate<br>&nbsp; -40 µl 50-150 µg protein in 50 mM K-phosphate – 5mM DTT – 1 mM EDTA, pH 7.4<br>&nbsp; -40 µl 200 mM K-phosphate, pH 7.4<br>&nbsp; -1 µl 1U/µl glucose 6-phosphate dehydrogenase<br>&nbsp; ( total vol. 91µl)<br>17. Incubate 15 min in +37ºC heat block <br>18. Add 11 µl of substrate ([3-14C]HMG-CoA + cold HMG-CoA; see below)<br>19. Incubate 90 min in +37ºC <br>20. Stop the reaction with 10 µl of 5M HCl<br>21. Add 1 µl (= 1 mg) of mevalonic acid lactone<br>22. Incubate 30 min in +37ºC <br>23. You can store the samples o/n in +4ºC.
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15. Use 150 µg of protein/assay. If there is not enough protein in 40µl, 50µg is enough.<br>16. Combine the following reagents on ice in 1.5 ml tubes. Final volume is 102µl. <br>&nbsp; -10 µl 25 mM NADP – 200 mM glucose 6-phosphate<br>&nbsp; -40 µl 50-150 µg protein in 50 mM K-phosphate – 5mM DTT – 1 mM EDTA, pH 7.4<br>&nbsp; -40 µl 200 mM K-phosphate, pH 7.4<br>&nbsp; -1 µl 1U/µl glucose 6-phosphate dehydrogenase<br>&nbsp; ( total vol. 91µl)<br>17. Incubate 15 min in +37ºC heat block <br>18. Add 11 µl of substrate ([3-14C]HMG-CoA + cold HMG-CoA; see below)<br>19. Incubate 90 min in +37ºC <br>20. Stop the reaction with 10 µl of 5M HCl<br>21. Add 1 µl (= 1 mg) of mevalonic acid lactone<br>22. Incubate 30 min in +37ºC <br>23. You can store the samples o/n in +4ºC.  
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'''TLC'''
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'''TLC'''  
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24. Centrifuge the samples 5 min with full speed (microcentrifuge)<br>25. Take 80 µl of the supernatant into a fresh tube<br>26. Pipette 75 µl on TLC plate (width 2 cm; 2 cm from the edges, 2 cm from the bottom, 1.5 cm between the samples)(Merck silica gel 60, cat. no. 1.05721.0001)<br>27. As a standard you can use 1 µl mevalonic acid lactone<br>28. Run the TLC in chloroform:acetone 1:1. It takes approximately 50 minutes – 1 hour.<br>29. Stain the plate with iodine. The sample is approximately the half-way up the plate.<br>30. Mark the samples and let the iodine evaporate <br>31. Scrape the sample of the area of 2.4 cm (width) x 1,5 cm (height) to scintillation tube (with the help of methanol) <br>32. Analyze 14C radioactivity by liquid scintillation counting<br><br>'''Solutions'''
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24. Centrifuge the samples 5 min with full speed (microcentrifuge)<br>25. Take 80 µl of the supernatant into a fresh tube<br>26. Pipette 75 µl on TLC plate (width 2 cm; 2 cm from the edges, 2 cm from the bottom, 1.5 cm between the samples)(Merck silica gel 60, cat. no. 1.05721.0001)<br>27. As a standard you can use 1 µl mevalonic acid lactone<br>28. Run the TLC in chloroform:acetone 1:1. It takes approximately 50 minutes – 1 hour.<br>29. Stain the plate with iodine. The sample is approximately the half-way up the plate.<br>30. Mark the samples and let the iodine evaporate <br>31. Scrape the sample of the area of 2.4 cm (width) x 1,5 cm (height) to scintillation tube (with the help of methanol) <br>32. Analyze 14C radioactivity by liquid scintillation counting<br><br>'''Solutions'''  
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• 20mM Tris-HCl, 1mM EDTA pH 7.5<br>• 50 mM K-phosphate + 5 mM DTT + 1 mM EDTA pH 7.4.<br>&nbsp; -Because of the DTT the solution must be max two weeks old. <br>&nbsp; -Store the solution in dark +4ºC.<br>• 200mM K-phosphate, pH 7.4<br>• 25 mM NADP + 200 mM glucose 6-phosphate<br>• Glucose 6-phosphate dehydrogenase 1U/µl in water<br>• Substrate: [3-14C]HMG-CoA 10µCi/500µl, add 46µl HMG-CoA (10mg/ml in water)<br>• 5M HCl<br>• Mevalonic acid lactone 1mg/µl in water<br>• Chloroform:acetone 1:1
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• 20mM Tris-HCl, 1mM EDTA pH 7.5<br>• 50 mM K-phosphate + 5 mM DTT + 1 mM EDTA pH 7.4.<br>&nbsp; -Because of the DTT the solution must be max two weeks old. <br>&nbsp; -Store the solution in dark +4ºC.<br>• 200mM K-phosphate, pH 7.4<br>• 25 mM NADP + 200 mM glucose 6-phosphate<br>• Glucose 6-phosphate dehydrogenase 1U/µl in water<br>• Substrate: [3-14C]HMG-CoA 10µCi/500µl, add 46µl HMG-CoA (10mg/ml in water)<br>• 5M HCl<br>• Mevalonic acid lactone 1mg/µl in water<br>• Chloroform:acetone 1:1  
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'''Reagents'''
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'''Reagents'''  
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• NADP, Boehringer Mannheim, cat 128058<br>• D-Glucose-6-phosphate, dipotassium salt, Sigma G-7375 (1g)<br>• Glucose-6-phosphate dehydrogenase 250 units, Sigma G-8164<br>• 3-hydroxy-3-methyl[3-14C] glutaryl coenzymeA = [3-14C]HMG-CoA 10µCi/370 kBq 500µl, Amersham CFA 732<br>• DL-hydroxy-3-methyl-glutaryl coenzymeA, Sigma H-6132 (5mg)<br>• DL-Mevalonic acid lactone, Sigma M-4667 (1g)
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• NADP, Boehringer Mannheim, cat 128058<br>• D-Glucose-6-phosphate, dipotassium salt, Sigma G-7375 (1g)<br>• Glucose-6-phosphate dehydrogenase 250 units, Sigma G-8164<br>• 3-hydroxy-3-methyl[3-14C] glutaryl coenzymeA = [3-14C]HMG-CoA 10µCi/370 kBq 500µl, Amersham CFA 732<br>• DL-hydroxy-3-methyl-glutaryl coenzymeA, Sigma H-6132 (5mg)<br>• DL-Mevalonic acid lactone, Sigma M-4667 (1g)  
<br>'''Reference<br>'''Wilce PA, Kroon PA (1992). In Lipoprotein Analysis – a Practical Approach (Converse CA, Skinner ER, eds.) Oxford Univ. Press, Oxford,<br>pp. 207-211.<br>
<br>'''Reference<br>'''Wilce PA, Kroon PA (1992). In Lipoprotein Analysis – a Practical Approach (Converse CA, Skinner ER, eds.) Oxford Univ. Press, Oxford,<br>pp. 207-211.<br>
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[[Category:Conventional lipid analysis]]

Revision as of 11:58, 21 October 2008

HMG-CoA-reductase -assay

1. You need a 10 cm dish of cells (60-240µg of protein of total membranes). Remember replicate samples.
2. Grow cells ~ 40 h in lipoprotein deficient medium
3. Keep the cells on ice. Wash cells twice with cold PBS
4. Collect cells by scraping in 5 ml PBS and wash the dish again with 2 ml of PBS. Move cells (and washes) to 15 ml Falcon tubes.
5. Pellet cells 2000 x g 5 min + 4ºC. You can freeze the cell pellets in -70ºC.
6. Lyse cells in 1 ml of 20mM Tris-HCl + 1 mM EDTA pH 7.5. Enhance the lysis by passing the lysate 20 times through a 21G syringe needle.
7. Transfer the lysates to ultracentrifuge tubes. You can increase the volume of the lysates with 1 ml of lysis buffer if necessary.
8. Ultracentrifuge 1 h at 100 000 x g in +4ºC to collect total membranes.
9. Discard the supernatant and either freeze the pellets in -70ºC or continue with protein assay and do the HMG CoA reductase assay during the same day

Determining the protein concentration
10. Resuspend the membrane pellets in 150 µl of cold 50 mM K-phosphate + 5 mM DTT (fresh) + 1 mM EDTA (difficult to resuspend)
11. Protein assay can be done with BIO-RAD DC protein assay –kit.
12. Dilute the samples (just the amount you need for the protein assay) 1:5 in water, because 5mM DTT disturbs the protein assay.
13. Dilute also the standards so that you get them in 1/5 x resuspension buffer.
14. Vortex the samples well before taking a sample for the assay

Assay

15. Use 150 µg of protein/assay. If there is not enough protein in 40µl, 50µg is enough.
16. Combine the following reagents on ice in 1.5 ml tubes. Final volume is 102µl.
  -10 µl 25 mM NADP – 200 mM glucose 6-phosphate
  -40 µl 50-150 µg protein in 50 mM K-phosphate – 5mM DTT – 1 mM EDTA, pH 7.4
  -40 µl 200 mM K-phosphate, pH 7.4
  -1 µl 1U/µl glucose 6-phosphate dehydrogenase
  ( total vol. 91µl)
17. Incubate 15 min in +37ºC heat block
18. Add 11 µl of substrate ([3-14C]HMG-CoA + cold HMG-CoA; see below)
19. Incubate 90 min in +37ºC
20. Stop the reaction with 10 µl of 5M HCl
21. Add 1 µl (= 1 mg) of mevalonic acid lactone
22. Incubate 30 min in +37ºC
23. You can store the samples o/n in +4ºC.

TLC

24. Centrifuge the samples 5 min with full speed (microcentrifuge)
25. Take 80 µl of the supernatant into a fresh tube
26. Pipette 75 µl on TLC plate (width 2 cm; 2 cm from the edges, 2 cm from the bottom, 1.5 cm between the samples)(Merck silica gel 60, cat. no. 1.05721.0001)
27. As a standard you can use 1 µl mevalonic acid lactone
28. Run the TLC in chloroform:acetone 1:1. It takes approximately 50 minutes – 1 hour.
29. Stain the plate with iodine. The sample is approximately the half-way up the plate.
30. Mark the samples and let the iodine evaporate
31. Scrape the sample of the area of 2.4 cm (width) x 1,5 cm (height) to scintillation tube (with the help of methanol)
32. Analyze 14C radioactivity by liquid scintillation counting

Solutions

• 20mM Tris-HCl, 1mM EDTA pH 7.5
• 50 mM K-phosphate + 5 mM DTT + 1 mM EDTA pH 7.4.
  -Because of the DTT the solution must be max two weeks old.
  -Store the solution in dark +4ºC.
• 200mM K-phosphate, pH 7.4
• 25 mM NADP + 200 mM glucose 6-phosphate
• Glucose 6-phosphate dehydrogenase 1U/µl in water
• Substrate: [3-14C]HMG-CoA 10µCi/500µl, add 46µl HMG-CoA (10mg/ml in water)
• 5M HCl
• Mevalonic acid lactone 1mg/µl in water
• Chloroform:acetone 1:1

Reagents

• NADP, Boehringer Mannheim, cat 128058
• D-Glucose-6-phosphate, dipotassium salt, Sigma G-7375 (1g)
• Glucose-6-phosphate dehydrogenase 250 units, Sigma G-8164
• 3-hydroxy-3-methyl[3-14C] glutaryl coenzymeA = [3-14C]HMG-CoA 10µCi/370 kBq 500µl, Amersham CFA 732
• DL-hydroxy-3-methyl-glutaryl coenzymeA, Sigma H-6132 (5mg)
• DL-Mevalonic acid lactone, Sigma M-4667 (1g)


Reference
Wilce PA, Kroon PA (1992). In Lipoprotein Analysis – a Practical Approach (Converse CA, Skinner ER, eds.) Oxford Univ. Press, Oxford,
pp. 207-211.

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