Plant Physiology (Biology 327) - Dr. Stephen G. Saupe; College of St. Benedict/ St. John's University; Biology Department; Collegeville, MN 56321; (320) 363 - 2782; (320) 363 - 3202, fax; ssaupe@csbsju.edu |
Measuring Anthocyanin Content in Isolated Red Cabbage Protoplasts
Background:
In this exercise we will spectrophotometrically determine the
anthocyanin content of the red cabbage protoplasts in an acidified methanol extract. This
calculation is based on the Beer-Lambert Law: A = ecl [where e = molar extinction
coefficient of anthocyanin which is 38,000 M-1cm-1 (Hrazdina et al, 1982); c
= concentration in M; l = pathlength in cm, which is usually 1.0 cm (or 1.2 cm in a Spec
20)]. For more on measuring anthocyanin concentrations,
click here.
Questions: What is the anthocyanin content of a red cabbage protoplast? Is the concentration of anthocyanin similar to that of red grape protoplasts (1.67 pmol/protoplast, 0.097 M; Moskowitz and Hrazdina, 1981)?
Hypothesis: The anthocyanin content of red cabbage will be approximately 2.0 pmol/protoplast or 100 mM.
Protocol:
Data:
Table 1: Absorbance and anthocyanin content in a 0.1 ml sample of a partially purified red cabbage protoplast suspension in acidified methanol. | |||||
Sample |
A553 |
[Anthocyanin] (M) (in cuvette) | [Anthocyanin] (M) in sample (remember dilution) | [Anthocyanin] (pmol/protoplast) | [Anthocyanin] in a protoplast (M) |
1 |
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2 |
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3 |
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4 |
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5 |
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6 |
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7 |
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8 |
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9 |
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10 |
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Mean (+/- std dev) |
Table 2: Summary Table | |
Mean Protoplast Volume (pl) (from our ocular micrometer data; previous lab) | |
Mean [protoplast] (protoplasts ml-1; hemocytometer data) | |
Mean [anthocyanin] (pmol/protoplast) | |
Mean [anthocyanin] (M) |
Analysis and Conclusions:
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Last updated:
01/07/2009 � Copyright by SG
Saupe