Abstract
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Determining Protein content of Drosophila
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BCA protein assay will help find out the protein concentration in flies as the more Cu+ ions there is, the more intense the purple will be in the solution. It will allow the spectrophotometry to determine the concentration of the solutions. The results were gained by using six different Eppendorf tubes which are labelled 1600, 800, 400, 200, 100 and 0 g/ml. 50 l of the 1600 g/ml is added to 1600 and then another to 800 g/ml. Distilled water is added to the rest of them and 50 l of the protein solution. The flies from the square Eppendorf tube is added to a weighed out Eppendorf tube. They are crushed with a blue pestle and homogenizing buffer is added. The results show that there is no statistical difference between the two different homogenates. ‘Table 4’ shows the absorbance’s for them. Overall, they show that there is no statistical difference between them and the protein content of the Drosophila is shown in ‘Figure 1’.
Introduction
The practical is to determine the protein content of male Drosophila flies. In order to determine how much protein content there is in male Drosophila flies, we need to use BCA (bicinchoninic acid) assay. This will allow us to find out the protein concentration in the flies. It turns the solution into a purple-coloured product and it will allow it to absorb light at a wavelength of 562 nm (G-Biosciences, 2018). The BCA assay works by reducing the Cu2+ to Cu+1 which results in the purple colour solution and it can help reduce the variability that is caused by the compositional differences in protein (Fanglian, 2018). It is used because of the advantage that it can reduce the variability in the compositional differences in protein. The more protein there is, the more intense the purple in the solution will be. Spectrophotometry follows the Beer-Lambert Law which is where the absorbance of a solution at a specific wavelength (A) is directly proportionate to that of the concentration of the absorbing molecule (C). The more protein present, the more Cu+ complexes with the BCA and this will mean more absorption. Protein in the flies will react with the BCA reagent and it will give an absorbance that might be able to be read off the standard curve.
Methods
Standards
Label the Eppendorf tube with the dilution concentration that it will contain including a zero concentration Eppendorf tube. Label them from 1600, 800, 400, 200, 100 and 0 g/ml. Pipette 50 l of the 1600 g/ml protein solution which is Bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) into the 1600 g/ml labelled Eppendorf tube. Pipette another 50 l into the 800 g/ml Eppendorf tube. Make sure when pipetting into Eppendorf tube to take them out of the tube rack to ensure that you can see the tip clearly. Discard the pipette tip to avoid contamination. Using a fresh pipette tip, add 50 l of water into the other Eppendorf tubes (0, 100, 200, 400) and use the same tip to add 50 l into the 800 g/ml Eppendorf tube. Using the same tip still mix carefully inside the 800 g/ml tube and transfer 50 l of the solution. Transfer this to the 400 g/ml Eppendorf tube and then to the rest. This is known as a serial 1 in 2 dilutions. Remove 50 l out from the 100g/ml Eppendorf tube since the rest of them have 50 l but it has 100 l.
Fly samples
Using the weighed tube and a tube that has a square on it, transfer the flies that come from the square Eppendorf tube into the Eppendorf that has been weighed and labelled. Add 100 l of homogenizing buffer and crush the flies with a plastic pestle. After a minute of crushing, add another 300 l of the buffer and crush until there are no large bits that are visible. After 2 minutes add another 200 l of the buffer, close the lid and vortex for 30 seconds. After 5 minutes, centrifuge the homogenate at 3000g for 3 minutes. Remove it from the centrifuge and transfer 200 l of the supernatant to a fresh Eppendorf tube. Transfer 50 l into three separate Eppendorf tubes. Label them appropriately.
BCA Assay
Pipette 1000 ml BCA reagent (Thermo Scientific, Rockford, IL, USA) and add it to all the samples and place them into a 60oC waterbath for 30 minutes to incubate. After the 30 minute are done, immediately read the absorbance’s of all the solutions using the spectrophotometer. Using the zero tube to calibrate the spectrophotometer. Set the wavelength to 590 nm. Take a plastic cuvette and pour the contents of the Eppendorf tubes into it. Read then record the absorbance of them all. Including the unknowns. As the unknowns were above 1, it was diluted by adding 200 l of distilled water and 800 l of the absorbance replicate.
Results
Figure 1: Shows the standard curve for the protein concentration, this will help determine the protein concentration in the unknown sample. Calculated the protein concentration by dividing the average of the three absorbance replicates by 0.0027. 0.283 / 0.0027 =104.81
Figure 2: This shows whether is a statistical difference between the mass of the two different fly homogenates
Figure 3: This shows whether is a statistical difference between the protein percentage of the two different fly homogenates
Cross
Weight (mg)
Protein Percentage (%)
Mean
3.2
11.7
Standard Deviation
0.6
4.5
Table 1: Shows the mean and standard deviation of weight and protein percentage in cross Eppendorf tubes.
Square
Weight (mg)
Protein Percentage (%)
Mean
2.9
10.6
Standard Deviation
0.6
4.8
Table 2: Shows the mean and standard deviation of weight and protein percentage in the square Eppendorf tubes.
g/mL
1600
800
400
200
100
0
Absorbance
Too high
1.351
0.958
0.758
0.420
0
Table 3: Showing the protein concentration BSA standard dilution absorbance readings.
X5
Absorbance replicate 1
0.057
0.285
Replicate 2
0.056
0.281
Replicate 3
0.057
0.285
Table 4: Showing the fly homogenate absorbance readings. Multiply the replicates by 5 due to them being a 1 in 5 dilution. Average of them all will equal 0.283.
Discussion
‘Figure 2’ and ‘Figure 3’ show that there is no statistical difference between the two different homogenates. This is due to error bars overlapping each other in the bar graphs. There are some disadvantages with the BCA protein assay such as that compared to other assays such as Bradford assay, it is susceptible to some interference by chemicals present in the protein (Fanglian, 2018). Bradford assay could be used which is cheap but it works more quick and easy than BCA protein assay. It relies on direct binding to protein samples and it is compatible with a wide range of components (G-Biosciences, 2018). This means that the Bradford assay is better than BCA protein assay as unlike the BCA protein assay it is compatible with a wider range of components. This means that Bradford assay would have been best to use than using BCA protein assay. ‘Table 1’ and ‘Table 2’ are showing the standard deviation which is meant to show how much they differ from the mean value. T-test can’t be used for these results as there are too many samples, t-test is usually used when there is a small sample size.
References
Fanglian, H. (2018). BCA (Bicinchoninic Acid) Protein Assay —BIO-PROTOCOL. [online] Bio-protocol.org. Available at: https://bio-protocol.org/bio101/e44 [Accessed 12 Nov. 2018].
G-Biosciences. (2018). Bicinchoninic Acid (BCA) Protein Assay. [online] Available at: https://www.gbiosciences.com/Protein-Research/Bicinchoninic-Acid-BCA-Protein-Assay [Accessed 12 Nov. 2018].
G-Biosciences. (2018). Is Your BCA Protein Assay Really the Best Choice?. [online] Available at: https://info.gbiosciences.com/blog/alternative-bca-protein-assay [Accessed 12 Nov. 2018].
BOVINE SERUM ALBUMIN: Sigma-Aldrich, St. Louis, MO, USA.
BCA protein assay reagents A and B: Thermo Scientific, Rockford, IL, USA.
Raw Data
Fly symbol
Mass of flies
Protein conc
Total protein
Total protein
Protein
(Square or Cross)
(mg)
(g/ml)
(μg)
(mg)
(%)
CR
3.0
694.2
416.5
0.4
13.9
CR
3.4
324.4
194.6
0.2
5.7
CR
4.5
562.0
337.2
0.3
7.5
CR
4.1
457.1
274.3
0.3
6.7
CR
2.5
840.9
504.5
0.5
20.2
CR
2.6
642.2
385.3
0.4
14.8
CR
2.9
995.7
597.4
0.6
20.6
CR
2.1
527.2
316.3
0.3
15.1
CR
2.7
734.2
440.5
0.4
16.3
CR
2.1
688.3
413.0
0.4
19.7
CR
3.0
481.1
288.7
0.3
9.6
CR
2.8
327.3
196.4
0.2
7.0
CR
3.1
474.4
284.6
0.3
9.2
CR
2.5
537.8
322.7
0.3
12.9
CR
3.6
768.3
461.0
0.5
12.8
CR
3.2
515.2
309.1
0.3
9.7
CR
3.3
646.0
387.6
0.4
11.7
CR
3.4
593.3
356.0
0.4
10.5
CR
2.3
642.1
385.2
0.4
16.7
CR
2.7
284.2
170.5
0.2
6.3
CR
3.3
1019.5
611.7
0.6
18.5
CR
3.0
569.1
341.4
0.3
11.4
CR
2.2
607.8
364.7
0.4
16.6
CR
4.1
467.5
280.5
0.3
6.8
CR
3.0
508.7
305.2
0.3
10.2
CR
3.2
363.7
218.2
0.2
6.8
CR
3.4
318.3
191.0
0.2
5.6
CR
3.1
1118.7
671.2
0.7
21.7
CR
5.1
761.0
456.6
0.5
9.0
CR
4.3
984.2
590.5
0.6
13.7
CR
3.0
510.2
306.1
0.3
10.2
CR
3.9
838.3
503.0
0.5
12.9
CR
3.2
602.8
361.7
0.4
11.3
CR
3.9
1069.4
641.7
0.6
16.5
CR
3.7
621.3
372.8
0.4
10.1
CR
3.3
445.0
267.0
0.3
8.1
CR
2.0
795.2
477.1
0.5
23.9
CR
2.9
405.6
243.3
0.2
8.4
CR
3.5
470.8
282.5
0.3
8.1
CR
3.1
784.0
470.4
0.5
15.2
CR
3.4
494.8
296.9
0.3
8.7
CR
3.4
802.9
481.7
0.5
14.2
CR
3.6
397.7
238.6
0.2
6.6
CR
2.5
613.7
368.2
0.4
14.7
CR
2.9
644.9
386.9
0.4
13.3
CR
3.2
716.0
429.6
0.4
13.4
CR
3.6
792.3
475.4
0.5
13.2
CR
3.4
594.6
356.7
0.4
10.5
CR
2.6
264.3
158.6
0.2
6.1
CR
4.4
786.8
472.1
0.5
10.7
CR
3.1
613.3
368.0
0.4
11.9
CR
4.0
235.6
141.3
0.1
3.5
CR
3.9
156.9
94.1
0.1
2.4
CR
3.0
556.3
333.8
0.3
11.1
CR
3.4
327.9
196.7
0.2
5.8
CR
3.5
722.7
433.6
0.4
12.4
CR
2.7
861.5
516.9
0.5
19.1
CR
3.3
805.7
483.4
0.5
14.6
CR
3.0
540.0
324.0
0.3
10.8
CR
3.7
500.0
300.0
0.3
8.1
CR
2.7
433.1
259.9
0.3
9.6
CR
3.4
512.6
307.6
0.3
9.0
CR
2.8
472.7
283.6
0.3
10.1
CR
2.7
575.0
345.0
0.3
12.8
CR
3.0
817.7
490.6
0.5
16.4
Weight
Protein Percentage
Mean
3.2
11.7
SD
0.6
4.5
SQ
2.7
544.4
326.7
0.3
12.1
SQ
2.7
687.6
412.6
0.4
15.3
SQ
2.8
524.4
314.6
0.3
11.2
SQ
2.4
569.0
341.4
0.3
14.2
SQ
2.0
589.2
353.5
0.4
17.7
SQ
3.5
645.0
387.0
0.4
11.1
SQ
3.0
597.9
358.8
0.4
11.9
SQ
2.9
578.6
347.1
0.3
12.0
SQ
2.2
367.1
220.2
0.2
10.0
SQ
2.8
252.2
151.3
0.2
5.4
SQ
3.9
1051.3
630.8
0.6
16.2
SQ
2.7
573.2
343.9
0.3
12.7
SQ
3.7
1113.8
668.3
0.7
18.1
SQ
2.7
1013.5
608.1
0.6
22.5
SQ
3.1
532.8
319.7
0.3
10.3
SQ
3.4
536.7
322.0
0.3
9.5
SQ
2.3
306.0
183.6
0.2
8.0
SQ
2.4
861.8
517.1
0.5
21.5
SQ
3.1
620.0
372.0
0.4
12.0
SQ
2.2
496.5
297.9
0.3
13.5
SQ
2.3
636.9
382.1
0.4
16.6
SQ
3.0
351.5
210.9
0.2
7.0
SQ
3.7
439.4
263.6
0.3
7.1
SQ
3.0
300.0
180.0
0.2
6.0
SQ
3.2
463.2
277.9
0.3
8.7
SQ
3.4
702.9
421.7
0.4
12.4
SQ
2.4
48.0
28.8
0.0
1.2
SQ
4.1
707.4
424.5
0.4
10.4
SQ
2.6
300.0
180.0
0.2
6.9
SQ
2.4
493.5
296.1
0.3
12.3
SQ
2.5
292.5
175.5
0.2
7.0
SQ
2.7
352.6
211.6
0.2
7.8
SQ
2.8
427.8
256.7
0.3
9.2
SQ
4.0
281.3
168.8
0.2
4.2
SQ
2.1
101.3
60.8
0.1
2.9
SQ
2.2
784.3
470.6
0.5
21.4
SQ
2.1
101.3
60.8
0.1
2.9
SQ
2.7
341.6
205.0
0.2
7.6
SQ
3.7
650.6
390.3
0.4
10.5
SQ
2.7
330.6
198.3
0.2
7.3
SQ
2.3
655.2
393.1
0.4
17.1
SQ
2.8
513.7
308.2
0.3
11.0
SQ
3.5
353.3
212.0
0.2
6.1
SQ
4.5
643.3
386.0
0.4
8.6
SQ
2.9
353.3
212.0
0.2
7.3
SQ
4.3
545.24
327.1
0.3
7.6
SQ
2.8
490.9
294.5
0.3
10.5
SQ
2.9
393.1
235.9
0.2
8.1
SQ
2.1
548.9
329.4
0.3
15.7
SQ
2.7
516.8
310.1
0.3
11.5
SQ
2.0
77.1
46.3
0.0
2.3
Weight
Protein Percentage
Mean
2.9
10.6
SD
0.6
4.8
T-Test(SQ/CR)
0.2
