Anthrone cellulose quantification

Updated version of cellulose quantification method described by Updegraff in 1969, for use in 1.5 mL tubes. Based on a protocol by Stewart Gillmor.

Materials

• Spectrophotometer (e.g. Jenway Genova Life Science Analyzer)
• Microbalance (e.g. Mettler UMT 2)
• Tin capsules (weighing vessels)
• 1 mL quartz cuvettes
• Acetone
• Millipore water

Solutions

• Anthrone solution: 100 mg anthrone (CAS 90-44-8) in 10 mL concentrated sulfuric acid. Must be prepared fresh daily
• Acetic nitric reagent: 150 mL 80% acetic acid plus 15 mL concentrated nitric acid.
• 10 mM glucose stock solution: 180.16 mg glucose in 100 mL ddH20

Procedure

1. Collect 10 Arabidopsis seedlings in 70% ethanol. Warm them at 70º C for one hour.
2. Take off the ethanol, replace it with more 70% ethanol and warm at 70º C for another 45 minutes.
3. Remove ethanol, add 1 mL acetone, and let sit for 10 minutes.
4. Remove acetone and let samples dry overnight (or speed-vac, if necessary).
5. Mass seedlings on an analytical balance.
6. Add 1 mL of acetic nitric reagent, heat at 98º C for 30 minutes.
7. Spin seedlings down at 14000 rpm for 10 minutes, then remove most of the supernatant, being careful not to lose any of the tissue.
8. Add 1 mL of ddH2O and invert. Spin down for another 10 minutes at 14000 rpm.
9. Remove half of the supernatant, add 1 mL acetone, and spin for 5 minutes.
10. Remove 1 mL of the supernatant, add 1 mL acetone, spin for another 5 minutes.
11. Remove as much of the supernatant as possible without losing tissue, then dry overnight or speed vac.
12. Add 100 µL 67% H2SO4. Cover, mix well on vortexer.
13. Mix 400 µL ddH2O with this solution, and then add 1 mL of anthrone solution (to tubes on ice). Boil on heat block for 5 minutes.
14. Transfer samples back to ice, then fill a glass or quartz cuvette with the blank (no glucose) solution.
15. Blank machine, read absorbance of all samples at 620 nm, find glucose concentration with standard curve. Wash the cuvette out with both deionized water and acetone each time to prevent a white deposit from clouding the cuvette window.

Make a glucose standard curve: 3 replicates each of the following six solutions:

Total glucose (nmol): 50, 100, 150, 200, 300, 400

Volume of ddH2O (µL): 495, 490, 485, 480, 470, 460

Volume of 10 mM Glc soln. (µL): 5, 10, 15, 20, 30, 40

Add 1 mL anthrone solution to these 500 µL volumes, read OD at 620 nm.

Notes on procedure

1. Polypropylene 1.5 mL tubes and pipette tips are stable to concentrated sulfuric acid, though the use of disposeable polystyrene cuvettes is not recommended (S. Gillmor, personal communication). Tubes should be sealed with cap-locks for all heating steps, especially ones involving acid.
2. Massing dry tissue directly in 1.5 mL tubes is not recommended, as we found that the resulting static electricity caused the microbalance we used to drift, sometimes quite significantly. Transferring tissue from small aluminum weighing vessels to tubes, and then finding the mass transferred by difference yielded much more stable and objective dry mass values. The microbalance values agreed with the values from less precise balances to within their uncertainty. Samples should be handled with tweezers to avoid local air currents from heating.
3. The mixing of sulfuric acid and water is highly exothermic, and is capable of driving the reaction of carbohydrate with anthrone. Accordingly, tubes should be kept on ice upon addition of anthrone solution in step 13, and then all transferred together to a heatblock as quickly as possible to allow the reaction to proceed to the same extent in all tubes (Scott and Melvin, 1953). If multiple batches of tubes are heated in a single day, it is quite important to control the temperature of the heat block in addition to heating time, as failing to control for this results in large absorbance differences between identical standards (not shown). For a five minute boiling time, approximately 75-175 nmol glucose corresponds to the linear range with absorbances between 0.4 and 0.9 that is ideal for spectrophotometric quantification, so tissue samples should have total glucose levels approximately in this range.
4. Scott and Melvin (Scott and Melvin, 1953), who achieved standard deviations of 0.48%, noted that variation between blank samples from minute carbohydrate contamination seems to be the main source of uncertainty in the spectrophotometric assay. Our blanks for a given day generally differed by less than 0.01 absorbance units, indicating small amount of instrument drift. In one run we used a method blank, performing all the extraction steps on an empty tube, and its final absorbance was essentially the same as the standard blank, providing confidence that our samples were not contaminated by stray polysaccharides during the extraction steps.
5. If solution heated in step 13 is insufficiently acidic, a white precipitate forms, preventing collection of absorbance data. If the solution is heated much longer than 5 minutes it will turn brown, and the absorbance at 620 nm will no longer be linear with glucose concentration.
6. There is danger of losing tissue in the various wash steps, especially step 12, because the acetic-nitric reagent treatment turns tubes yellow and the pelleted tissue covered with supernatant is very difficult to see. This problem may have been especially acute when we used an old centrifuge that may not have achieved its nominal maximum speed, causing the seedlings to not pellet properly.