Determining Protein Length from Western Blot

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Discussion Overview

The discussion revolves around determining the length (mass) of proteins A and B from a Western blot image, focusing on the graphical analysis of protein migration in an SDS-PAGE gel.

Discussion Character

  • Technical explanation
  • Exploratory

Main Points Raised

  • Some participants suggest that the mass scale is established using a protein ladder, from which the masses of unknown proteins can be inferred.
  • One participant explains that the distance migrated by proteins in an SDS-PAGE gel correlates with the logarithm of their molecular weight, proposing a method to plot these distances against their logarithmic weights to create a best-fit line for estimation.
  • Another participant mentions a different approach, indicating the need to measure distances from the top of the well rather than the bottom, raising a question about how to plot these distances and molecular weights step-by-step.

Areas of Agreement / Disagreement

Participants express differing views on the method of measuring distances for plotting, indicating that multiple approaches exist without a consensus on the best method.

Contextual Notes

There are unresolved details regarding the specific steps for plotting distances and molecular weights, as well as the implications of measuring from different reference points on the gel.

raymanray
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Hello Forum,

please take a look at this image

http://imageshack.us/photo/my-images/98/0bigg.jpg/

It shows a Western blot of an extract of proteins.

My question is how can you determine the length of both proteins A and B graphically?

Could you please help me?
 
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raymanray said:
Hello Forum,

please take a look at this image

http://imageshack.us/photo/my-images/98/0bigg.jpg/

It shows a Western blot of an extract of proteins.

My question is how can you determine the length of both proteins A and B graphically?

Could you please help me?

ITYM 'mass' of the protein. IIRC, the mass scale is set using the ladder (the set of spots on the left), and the masses of unknown proteins is then read off the scale.
 
The distance migrated by proteins in an SDS-PAGE gel is related to the logarithm of the molecular weight. So, you can measure the distance migrated from the bottom of the well for each of the protein standards in lane 1, plot these distances versus the logarithms of their molecular weights, and find the best-fit line. This best-fit line can then be used to estimate the molecular weights of your samples in lane 2.
 
Thank you for your answers.
 
Ygggdrasil said:
The distance migrated by proteins in an SDS-PAGE gel is related to the logarithm of the molecular weight. So, you can measure the distance migrated from the bottom of the well for each of the protein standards in lane 1, plot these distances versus the logarithms of their molecular weights, and find the best-fit line. This best-fit line can then be used to estimate the molecular weights of your samples in lane 2.

Thank you for your answer.

I read on other websites that I have to measure the distance from the side above from where the proteins started.

How can I plot the distances and their molecular weights? Can you tell me step-by-step please?
 

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