Académique Documents
Professionnel Documents
Culture Documents
TECHNICAL BULLETIN
Results
Calculations Concentration of Trolox Equivalents
The background for the assays is the value obtained for Sa/Sv = C
the 0 (blank) Trolox Standard. Correct for the
background by subtracting the 0 (blank) standard value where:
from all readings. Background values can be significant Sa = Trolox equivalent of unknown sample well
and must be subtracted from all readings. Use the (nmole) from standard curve
values obtained from the appropriate Trolox Standards Sv = Sample volume (L) added into the wells
to plot a standard curve. C = Concentration of antioxidant in sample (nmole/L
Note: A new standard curve must be set up each time or mM Trolox equivalents)
the assay is run.
Sample Calculation
Using the corrected measurement, Trolox equivalents Amount of antioxidant = 4.84 nmole
of the sample may be determined from the standard (from Trolox standard curve)
curve.
Sample volume (Sv) = 50 L
References
1. Valko, M. et al., Free radicals and antioxidants in
normal physiological functions and human disease.
Int. J. Biochem. Cell Biol., 39(1), 4484 (2006).
2. Sies, H., Oxidative stress: oxidants and
antioxidants. Exp. Physiol., 82(2), 291295 (1997).
4
Troubleshooting Guide
Problem Possible Cause Suggested Solution
Ice Cold Assay Buffer Assay Buffer must be at room temperature
Omission of step in procedure Refer and follow Technical Bulletin precisely
Assay not working
Plate reader at incorrect wavelength Check filter settings of instrument
Type of 96 well plate used For colorimetric assays, use clear plates
Use the Assay Buffer provided or refer to
Samples prepared in different buffer
Technical Bulletin for instructions
Repeat the sample homogenization,
Cell/Tissue culture samples were
increasing the length and extent of
incompletely homogenized
homogenization step.
Samples with erratic
Samples used after multiple freeze-thaw Aliquot and freeze samples if samples will be
readings
cycles used multiple times
Presence of interfering substance in the
If possible, dilute sample further
sample
Use of old or inappropriately stored Use fresh samples and store correctly until
samples use
Thaw all components completely and mix
Improperly thawed components
gently before use
Use of expired kit or improperly stored Check the expiration date and store the
Lower/higher reagents components appropriately
readings in samples Allowing the reagents to sit for extended Prepare fresh Master Reaction Mix before
and standards times on ice each use
Refer to Technical Bulletin and verify correct
Incorrect incubation times or temperatures
incubation times and temperatures
Incorrect volumes used Use calibrated pipettes and aliquot correctly
Thaw and resuspend all components before
Use of partially thawed components
preparing the reaction mix
Pipetting errors in preparation of standards Avoid pipetting small volumes
Prepare a Master Reaction Mix whenever
Pipetting errors in the Reaction Mix
possible
Non-linear standard
Air bubbles formed in well Pipette gently against the wall of the tubes
curve
Standard stock is at incorrect Refer to the standard dilution instructions in
concentration the Technical Bulletin
Recheck calculations after referring to
Calculation errors
Technical Bulletin
Substituting reagents from older kits/lots Use fresh components from the same kit
Samples measured at incorrect
Check the equipment and filter settings
wavelength
Unanticipated results Samples contain interfering substances If possible, dilute sample further
Sample readings above/below the linear Concentrate or dilute samples so readings
range are in the linear range
VI,KVG,LS,MAM 09/141
2014 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other
countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their
particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at
www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.