Changes In Hemolymph During M. sexta
Development
--Introduction--
There are several options for this series of experiments.
The first is simply to collect
hemolymph from a larva and a pupa and to make
comparisons in total protein concentration and/or protein
composition between the two life stages. Another option is
to obtain larvae of different ages and to perform
comparative analyses on their hemolymph.
The concentration of total protein in the hemolymph
samples can be determined by a Bradford
assay. This technique involves reacting the hemolymph
sample with a dye that binds protein. A reading is then made
of the absorbance of the sample using a spectrophotometer.
The concentration of total protein in the sample is
determined by comparing the absorbance of the hemolymph
sample against a standard curve. In the standard curve,
known amounts of protein are reacted with the dye and the
absorbance is taken of the samples. The amount of protein is
then plotted against the absorbance.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
is a technique used to separate proteins in a mixture by
molecular weight. The proteins in the hemolymph sample are
denatured and coated with a negatively charged detergent.
The sample is then loaded into a matrix. When an electric
current is applied to the matrix, the proteins migrate to
the positive pole with the smallest proteins migrating the
fastest. The position of the proteins in the matrix is then
made visible using a dye that stains the proteins.
For either of these experiments, the quantitative data
from an entire class can be pooled for statistical analysis.
If several of the experiments are performed, students can
integrate data across the experiments. For instance, is a
change in the total protein concentration consistent with
the results observed on the SDS-PAGE?
Protocols:
Collection of M. sexta
hemolymph.
Determination of protein
concentration of M. sexta hemolymph.
SDS-polyacrylamide gel
electrophoresis of M. sexta hemolymph.
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