Changes In Hemolymph During the Antibacterial
Response of M. sexta
Protocol: Quantitative Determination of
Quantitative estimation of the total protein content of a
sample is frequently necessary in cell physiological and
biochemical studies. Several methods of determining the
total protein content of a sample have been developed and
widely used during this century. One of the simplest and
most sensitive is the "Bradford" assay, which was introduced
in the mid-1970s. This assay is based on the binding
specificity of the dye Coomassie Brilliant Blue-G250 for
protein molecules but not for other cellular constituents.
This organic dye binds specifically to tyrosine side
The binding of the dye to protein shifts the peak
absorbance of the dye. Unbound Coomassie Blue absorbs light
maximally at a wavelength of 465 nm, while the absorption
maximum is at 595 nm when the dye is bound to protein. The
absorbance of light by the dye-protein complex at 595 nm is
proportional to the amount of protein bound (over a limited
range); i.e. there is a linear relationship between
absorbance and the total protein concentration of the sample
over a narrow range.
- bench diaper
- waste container (~ 250 ml plastic beaker)
- test tube rack with 30 test tubes
- 5 ml pipette; 1 10 ml pipette; 1 25 ml pipette
- pipette pump/bulb
- micropipettors P-200, P-20
- pipette tips for above
- distilled water in a microfuge tube
- plastic spectrophotometer cuvettes
- lab tissues
- methanol in a wash bottle
- Bradford Reagent (Bio-Rad
Protein Assay Dye Reagent catalog number 500-0006)
- Protein Standard Solution (1 mg/ml bovine serum
albumin dissolved in 0.1% SDS- keep on ice)
Coomassie blue (the dye in the Bradford Reagent),
phosphoric acid, and methanol are very harmful when
swallowed, absorbed through the skin or splashed in the
eyes. Phosphoric acid and methanol may be fatal if swallowed
or absorbed through the skin. Phosphoric acid is corrosive
to the eyes, skin, nose, mouth, and throat and may be fatal
if breathed in high concentrations. Methanol is an acute
poison when swallowed or breathed in high concentrations.
Symptoms of methanol poisoning include headache, vomiting,
dizziness, weakness, blurred vision. Methanol is extremely
corrosive to the eyes. Coomassie blue, phosphoric acid, and
methanol are components of the Bradford Reagent.
Gloves, protective glasses and lab coats should be worn
when working with these compounds. Coomassie blue will also
Preparation Of A Standard Curve
1) Complete the two columns in the table below. For the
column labeled 'ug of BSA' you should determine how many
total ug of BSA have been added to the tube. For the column
labeled 'concentration of BSA in ug/ul' you should determine
the final concentration of BSA.
µl of protein standard sol.
µl of dist. H2O
µg of BSA
concentration of BSA in
2) Then prepare 19 test tubes according to the Table
- a. Prepare triplicate test tubes for each of
the protein concentrations except the blank.
- b. First, transfer the water to all tubes by spotting
the water on the side of the tube approximately halfway
down the tube. It is important that the drop is low in
the test tube to allow mixing.
- c. Add the correct volume of BSA to the drop of water
in the order listed (From 10 to 100).
- d. Use the same pipette tip throughout these
transfers. When finished, dispose of the tip.
- e. Calculate the number of µg of BSA and the
concentration (in µg/µl) in each tube.
The total volume that you add to each tube should be 100
3) Add 5 ml of Bradford reagent to each test tube using a
glass pipette. Mix (vortex) the contents of each tube
briefly (2-3 sec.) using a vortex stirrer. Be careful not to
vortex so vigorously that the solution comes out of the
4) Allow the tubes to incubate 5 min. at room
temperature. What is room temperature?
5) Pour about 3 ml of the "blank" (no protein) into a
cuvette (about 2/3 full).
6) Check to be certain that the wavelength of the
spectrophotometer is set at 595 nm. Insert the cuvette
containing the blank and "zero" the spectrophotometer.
7) Pour the blank back into its test tube. Save the
blank for later use! Shake out the remaining fluid from
the cuvette, but DO NOT rinse the cuvette with distilled
8) Add about 2 ml from one of the "10" tubes into the
cuvette. This is the sample cuvette. Record the absorbance
in the Table 1 provided in the
9) After recording the absorbance reading in your lab
notebook, pour the contents of the sample cuvette back into
the test tube. Shake out remaining fluid from the cuvette,
but DO NOT rinse the cuvette with distilled water. Repeat
for the other "10" samples.
10) Similarly, determine and record the absorbances of
the remaining samples - in ascending order. Why is the order
11) Plot the standard curve. Place the independent
variable (µg/µl protein&emdash;one of the columns
you filled out in the table above) on the x-axis and the
dependent variable (absorbance) on the y-axis. Select a
scale values so that the slope of the line is approximately
1.0 (about 45 degrees).
12) Discard the used Bradford Assay reagent in the sink
with running water.
Determination Of The Protein Concentration Of Insect
1. Set up and label a set of triplicate test tubes for
each sample of hemolymph. To each tube, add 5 µl of the
appropriate hemolymph and 95 µl of water. This is a
2. Add 5 ml of the 1x Bradford dye reagent to each.
Vortex. Wait at least 5 min.
3. Use the blank from the standard curve assay to re-zero
4. Read and record the absorbance of the hemolymph
samples in Table 2 and record
your results on the board.
5. Extrapolate the concentration of the diluted hemolymph
from the standard curve. Determine the concentration of the
protein in the hemolymph by multiplying this value by
6. If the absorbance reading of a hemolymph sample is not
on the scale of the standard curve prepared previously, you
will need to dilute a small portion of the hemolymph
sample (not the Bradford Assay) by a known amount.
7. Data from an entire class may be pooled and a simple
statistical analysis (e.g. t-test) performed on the class
data to determine whether there is a significant difference
in protein concentration in hemolymph between samples. This
analysis can be done to compare bacteria-treated or
untreated insects or it can be done to compare larval and
Data Sheets for this
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