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Tobacco Hornworms


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Development (hemolymph)

Changes In Hemolymph During M. sexta Development

Protocol: Quantitative Determination of Proteins

Introduction

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 chains.

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.

 

Materials

  • 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
  • spectrophotometers
  • micropipettors P-200, P-20
  • pipette tips for above
  • distilled water in a microfuge tube
  • plastic spectrophotometer cuvettes
  • vortexer
  • lab tissues
  • thermometer
  • methanol in a wash bottle
  • Bradford Reagent (Bio-Rad Protein Assay Dye Reagent catalog number 500-0006) diluted 1:5)
  • Protein Standard Solution (1 mg/ml bovine serum albumin dissolved in 0.1% SDS- keep on ice)

 

Hazards:

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 stain clothing.

 

Experimental Protocols:

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.

tube label
µl of protein standard sol.
µl of dist. H2O
µg of BSA
concentration of BSA in µg/µl

blank
0
100

10
10
90

20
20
80

40
40
60

60
60
40

80
80
20

100
100
0

2) Then prepare 19 test tubes according to the Table above:

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 µl.

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 tube.

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 water.

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 Results section.

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 important?

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 Hemolymph

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 1/20 dilution.

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 the spectrophotometer.

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 20.

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 pupal stages.

 

Data Sheets for this protocol.

 

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March 1999