Protein electrophoresis algorithm

  • Thread starter Thread starter victorqed
  • Start date Start date
  • Tags Tags
    Algorithm Protein
Click For Summary

Discussion Overview

The discussion revolves around the implementation of an application for analyzing protein electrophoresis gels, specifically focusing on the accurate quantification of protein bands such as albumin and various globulins. Participants explore issues related to the accuracy of the software-generated results compared to known standard values.

Discussion Character

  • Technical explanation
  • Debate/contested
  • Experimental/applied

Main Points Raised

  • One participant reports discrepancies between the calculated protein percentages from their application and known standard values, particularly noting that albumin levels appear too low.
  • Another participant inquires about the nature of the reported values, seeking clarification on whether they are based on migration distances, molecular weights, or intensities.
  • A participant clarifies that the values are percentages derived from the total area of the gel image.
  • Concerns are raised about the accuracy of the correct values, questioning whether a standard solution with known concentrations was used for validation.
  • It is suggested that the detector may be saturating for the albumin band, potentially leading to underestimation of its intensity, and proposes diluting the sample or adjusting detector settings as possible solutions.
  • One participant asserts that the correct values are from a prepared solution by a specialized producer, claiming their accuracy.
  • Another participant emphasizes the need for a scanner capable of accurately detecting albumin levels, indicating that the current scanner may not be suitable.
  • Concerns are raised about differential dye binding and the impact of lighting conditions on the gel analysis, with suggestions for addressing pixel sensitivity issues in imaging.
  • A participant acknowledges the possibility of light affecting the results and expresses intent to investigate further.

Areas of Agreement / Disagreement

Participants express differing views on the source of the discrepancies in protein quantification, with some attributing it to the scanner's limitations and others considering potential issues with dye binding or lighting conditions. The discussion remains unresolved regarding the exact cause of the inaccuracies.

Contextual Notes

Participants mention various factors that could influence the accuracy of the measurements, including the type of staining used, the sensitivity of the scanner, and the potential for differential dye binding. These factors remain unverified and contribute to the uncertainty in the results.

victorqed
Messages
8
Reaction score
0
Hello

I'm trying to implement a application that will scan the electrophoresis gels and draw a graphic of the proteins: albumin, alpha1, alpha2, beta1, beta2 and gamma.
The problem is that the resulted graphic is not as it should be. Albumin percentage is too low and the rest of the proteins are too high. Also, looking at the my graphic I can see that albumin is very low.

I tried to make a comparative test with many applications that already exist on the web and all of them are giving me the same values as I calculated. But this values are wrong so it is clear that I'm missing something essential.

One of this applications is ImageJ(open source).
http://imagej.nih.gov/ij/
http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/

Example:
I have attached a scan of a normal control done on a Sebia gel.
Correct values are:
Albumin : 66,05%
Alpha1 : 2,47%
Alpha2 : 9,14%
Beta1 : 6,9%
Beta2 : 4,33%
Gamma : 11,11%

Values obtained with my app and ImageJ are:
Albumin: 46,76%
Alpha1 : 3,77%
Alpha2 : 15,05%
Beta1 : 11,78%
Beta2 : 5,36%
Gamma : 17,29%

How can I reproduce the correct values?
Thank you
 

Attachments

  • CN.jpg
    CN.jpg
    1.8 KB · Views: 576
Last edited:
Biology news on Phys.org
What values are you reporting? Migration distances? Calculated molecular weights? Intensities?
 
Values are in percents and are calculated as part of the total area of the resulting graphic.
I will edit the original post to add % sign.
 
So, you are measuring the intensity of each band in the gel, then dividing by the total intensity. How do you know your correct values are correct? Are you making up a standard solution with known concentrations of each protein? Is there a way to check whether you have made this standard solution correctly?

Another possibility is that the intensity from the albumin (the top band I presume), is saturating your detector, so your measurement is lower than the amount of protein present. Diluting the sample and running again may help (or changing the settings on your detector). How are you quantifying the intensity of each band? What type of staining are you using to visualize the proteins?
 
  • Like
Likes   Reactions: 1 person
The correct values are from a prepared solution, made by a specialized producer.
Solution has these concentrations of proteins from fabrication.
So the correct values are 100% correct.

You are right, the scanner is not specially made for electrophoresis and it might not be sensitive enough to see the peak of the Albumin.
I will try to fix the problem software by increasing the Albumin peak until I reach the target values.

If Albumin intensity is the problem than I should reach all protein values at the same time as I increase albumin.

Thank you :)
 
So, the scanner is the problem.
There is no complete software solution, I need a scanner that will see all the Albumin.
Where can I find one?
 
Have you eliminated the possibility of differential dye binding? Also if you aren't using a light box type of instrument, then you have all sorts of things to worry about. For example the lighting varying from place to place on the gel, as well as the inherent differences in the pixel sensitivities of the camera (or lens vignetting, dust etc). Some of these camera problems can be fixed easily, others not so much. You can google for something like flat-frame calibration to see what can be done with regard to the differences in the inherent pixel sensitivities.
 
  • Like
Likes   Reactions: 1 person
All solutions are standard, differential dye binding shouldn't be a problem but I will ask.
Light could be a new problem. :)