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Questions 61 - 71

Questions 61 - 71. ELMA design. 61. Buffer Making. What would be the most practical way of making the Tris solution Weigh out 2.4228 g of Tris, dissolve in 20 ml of water, then adjust the pH to 8.1 by adding concentrated HCl

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Questions 61 - 71

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  1. Questions 61 - 71 ELMA design

  2. 61. Buffer Making • What would be the most practical way of making the Tris solution • Weigh out 2.4228 g of Tris, dissolve in 20 ml of water, then adjust the pH to 8.1 by adding concentrated HCl • Dissolve 2.4228 g of Tris in 20 ml water, then adjust the pH to 8.1 by adding dilute HCl • Dissolve 2.4228 g of Tris in 10 ml water, then adjust the pH to 8.1 with HCl, then add water to 20 ml • Dissolve about 2.5 g of Tris in about 10 ml water, adjust the pH to 8.1 with HCl, then add water to give a volume equal to 20 x (weight taken/2.4228) • Accurately measure 20 ml water and adjust its pH to 8.1 with HCl, then add 2.4228 g of Tris

  3. 62. Using a Stock Solution • To achieve the above, how much of the stock acetyl-CoA solution (as recommended by your assistant) should you add to the cuvette? • 120 µl • 8.2 µl • 1.2 µl • 100 µl • 2.5 µl

  4. 63. Absorbance to nmol • If the absorbance change was 0.75 per min, how fast is the AMOUNT of yellow product increasing in the cuvette? • 75 µmol per min • 50 nmol per min • 5 nmol per min • 15 nmol per min • 15 µmol per min

  5. 64. Small Volume Additions • You decide that you need 50 mU of citrate synthase (CS) in the cuvette. What is the MOST PRACTICAL way of doing this? • Adding 0.025 µl of the stock CS directly to the cuvette • Adding 25 µl of the stock CS directly to the cuvette • Mixing 1 µl of the stock CS with 999 ul Tris buffer and then adding 25 ul of this diluted CS to the cuvette • Mixing 10 µl of stock CS with 990 ul Tris buffer and then adding 2.5 ul of this diluted CS to the cuvette • Adding 2.5 µl of the stock CS directly to the cuvette

  6. 65. Rapid Colour Change • What is NOT a plausible interpretation of these observations? • Something in the citrate synthase stock has a lot of free thiol groups • The acetyl CoA solution that you made up has partially degraded to give CoA • You have added a lot more citrate synthase than you thought • The DTNB spontaneously degrades at the pH of the Tris • Your acetyl CoA solution contains some free thiol groups

  7. The Time Course • After some trouble-shooting, you have convinced yourself that you can now make the citrate synthase assay work. • The scheme that you have come up with is to set up a cuvette containing 20 mM TRIS, 1 mM EDTA, 0.1 mM DTNB, 0.1 mM acetyl-CoA and 50 mU of citrate synthase (total volume of 1 ml). Then you mix the cuvette contents, put it in the spectrophotometer and zero its absorbance. Then you add 0.05 mM oxaloaceate and record the absorbance change with time. • Over the first 30 seconds, you find that the absorbance rises by about 0.35.

  8. 66. Limiting Factors To make this system measure acetyl-CoA, you replace the stock acetyl-CoA with a volume of an ‘unkonwn’ sample (eg, a tissue extract) • Which statement is the MOST LIKELY prediction of what will happen next? • The rate of increase in absorbance will remain constant for at least 10 minutes • The absorbance will plateau when the acetyl-CoA runs out • The rate will fall after about 5 minutes as the citrate synthase becomes degraded • The absorbance will fall as the all the DTNB gets used up • In less than five minutes all the oxaloacetate will run out

  9. 67. ELMA principles • What else do you have to do to make this system useful as an assay for acetyl‑CoA? • Ensure that oxaloacetate is never in excess • Ensure that DTNB is in excess • Ensure that citrate synthase does not drive the reaction to completion in less than 5 minutes • Ensure that the absorbance of the system plateaus above 2 • Ensure that the unknown acetyl-CoA is added in excess

  10. 68. Role of Unknown Chemical • How does the Triton X-100 and freezing/thawing help you get the most accurate results from the tissue samples? • It activates citrate synthase • It increases the release of citrate synthase from the mitochondrial matrix • It causes the hydrolysis of acetyl-CoA • It increases the release of metabolites from the cell • It increases the turbidity of the extract

  11. 69. Tissue Extracts • When you use the real tissue extract, you anticipate problems will occur. Which is NOT likely to be a cause of such problems?. • Tissue extracts can contain pigments that absorb in the visible range • Tissue extracts may contain CoA • Tissue extracts contain citrate synthase • The acetyl CoA concentration in the tissue extracts may be too low • Tissue extracts can be cloudy

  12. 70. Back Calculation • If the acetyl-CoA concentration of a sample of heart homogenate is found to be 50 µM, what is the concentration of acetyl-CoA in the tissue? • 1000 nmol g-1 • 50 nmol g-1 • 10 nmol g-1 • 5000 nmol g-1 • 100 nmol g-1

  13. 71. Spotting Good Data • What would give you the most confidence that your acetyl-CoA assay was working well? • Each tissue extract gives a different final result for [acetyl-CoA] • Different volumes of the same tissue extract plateau at the same absorbance • The final result for [acetyl-CoA] for each tissue extract is identical • For a particular tissue extract, the plateau absorbance is proportional to the amount of extract added • The absorbance of each assay plateaus at the same value

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