Solve Braford Assay: Why is My Answer 20X Different?

  • Thread starter tvtokyo
  • Start date
In summary, the conversation is about a question on Bradford Assay and calculating the protein concentration of a liver sample. One person provided their calculation and answered their own question, but their answer differed from the correct answer by a factor of 20. Another person pointed out that the difference is due to a dilution factor of 20 in the given sample. The final calculation and answer are confirmed to be correct.
  • #1
8
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Homework Statement



https://www.physicsforums.com/attachments/76905
https://www.physicsforums.com/attachments/76906

Homework Equations

3. The Attempt at a Solution [/B]
Hi I have a question on Braford Assay:
I am given this question:
I did the caluclation as follows:
y = 66.717x
Since given absorbance = 0.562
0.562 = 66.717x
x = 0.00842ug/ul
But the answers given is 0.168mg/ml
I do not know what I did wrongly? Can anyone please help? Thanks!
My answer and the correct answer differ by a factor of 20? Why is that so?
 
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  • #2
tvtokyo said:
My answer and the correct answer differ by a factor of 20?

0.562 is not absorbance on the original solution.
 
  • #3
Yes, I was trying to answer this question but when I press your links this time I am told I don't have right to access the page. But I remember when I saw it before there was something about an 0.05 ml sample - which is 1/20 of an ml. Could these occurrences of 1/20 be in any easy connected?
 
  • #4
I still don't get it though. Sorry. I will post the image again, Sorry for the trouble pal!
Anyway, even it is a factor of 20. My answer has a unit of ug/ul and the correct answer is in mg/ml so technically it is not a factor of 20??

upload_2014-12-26_10-21-24.png

upload_2014-12-26_10-21-47.png
 
  • #5
Please reread the last paragraph of the attached text, it clearly tells you why the difference is 20.

50+750+200=1000.
 
  • #6
I am suppose to find the protein concentration of the liver sample what do i do with the 1000ul ?
Is this correct?
Since x = 0.00842ug/ul and total volume of my assay --> 1000 ul
Then mass = 0.00842 * 1000 = 8.42 ug
Concentration of protein = 8.42ug/50ul (of protein in liver)
= 0.1684 ug/ul = 0.1684 mg/ml
 
  • #7
Correct.

50 μl of your sample were diluted 20 times to 1000μl.
 
  • #8
Thank you!
 

1. What is the Braford Assay and why is it used?

The Braford Assay is a method used to determine the concentration of protein in a given sample. It is commonly used in biochemical and biological research to measure the amount of protein present in a solution. This information can be used in a variety of applications, such as protein purification, quality control, and protein quantification.

2. How does the Braford Assay work?

The Braford Assay uses the principle of colorimetry, where a color change is produced in response to the presence of protein. This color change can be measured using a spectrophotometer, which then correlates to the concentration of protein in the sample. The assay also uses a standard curve, where known concentrations of protein are used to create a linear relationship between absorbance and protein concentration.

3. What are some common sources of error when performing a Braford Assay?

One common source of error is the use of incorrect volumes or dilutions of samples and reagents. Another source of error can be contamination of the sample with other substances that interfere with the assay. Inaccurate measurements and inconsistent technique can also contribute to errors in the results.

4. Why might my Braford Assay results be 20x different from expected?

There are several factors that can contribute to a significant difference in results from the expected value. It could be due to incorrect sample dilution, errors in measurement, inaccuracies in the standard curve, or contamination of the sample. It is important to carefully follow the protocol and troubleshoot any potential sources of error to ensure accurate results.

5. How can I improve the accuracy and reproducibility of my Braford Assay results?

To improve the accuracy and reproducibility of Braford Assay results, it is important to carefully follow the protocol and use precise and accurate measurement techniques. It may also be helpful to repeat the assay multiple times and calculate the average value to account for any potential outliers. Conducting a thorough analysis of potential sources of error and addressing them accordingly can also improve the accuracy of results.

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