Estimating size of unknown protein

[Gamewizard]

Hi all ,

I may have created a similar thread before but I am sorry I need help again and this is a bit diffrent to before.

 

 

I recently did a gel electrophoresis experiment, and now I need to find out the mass of the unknown protein sample. For that I have been given MW markers that were run alongside the DNA (TE) and the E1 which contains the unknown protein sample. So my question is that would I need to plot the migration of the

E1 fragments against the size of the MW marker on a semi-log paper to estimate the mass of the unknown protein sample?

 

There are total 7 bands shown in the gel image for the MW marker, and thier sizes are 97.2, 66, 44, 29, 20, 14.3, 6.5, and they have to be plotted on the x axis and the distance i know is going to go on the y axis. But I dont know how to plot it on the semi-log ? as the x axis goes from 0 to 10.

 

I am so confused can anyone please help me

[DeltaScience]

Well first one question? is it a protein or a gene? hehe because if you're runing a protein against a DNA marker it wont make any sense, althoug you could estimate the amount of a.a of your protein depending of the gene you're verifiyng but it´s harder.

 

I think you can use a scale for the marker, if you say that it goes from 0 to 10 then you could use an scale in wich 10 would be a 100 or something like that. for the size of your protein you should check something about linear regresion using the MW as the reference for creating the equation, but I dont think that you have to use a plot to estimate the size of your protein, since I cant tell you the answer you should also check about the markers and what they're used for =P.

 

I hope it was helpful. Good luck

[Schrödinger's hat]

Hi all ,

I may have created a similar thread before but I am sorry I need help again and this is a bit diffrent to before.

 

 

I recently did a gel electrophoresis experiment, and now I need to find out the mass of the unknown protein sample. For that I have been given MW markers that were run alongside the DNA (TE) and the E1 which contains the unknown protein sample. So my question is that would I need to plot the migration of the

E1 fragments against the size of the MW marker on a semi-log paper to estimate the mass of the unknown protein sample?

 

There are total 7 bands shown in the gel image for the MW marker, and thier sizes are 97.2, 66, 44, 29, 20, 14.3, 6.5, and they have to be plotted on the x axis and the distance i know is going to go on the y axis. But I dont know how to plot it on the semi-log ? as the x axis goes from 0 to 10.

 

I am so confused can anyone please help me

 

Biology isn't my strong point and I don't know all your jargon, so I'm going to have to make some assumptions. If any of these are wrong, then my explanation is probably wrong.

 

1) You've done some kind of test based on diffusion, treating/separating the DNA for some proteins and allowing them to diffuse through some paper/similar.

The DNA for each protein stayed intact (but separate from other genes) by some process which is arcane to me.

2) You have data sheets for these MW marker genes (ie. they're known molecules).

3) Everything diffused in the same way for the same amount of time, molecule size.

Very crudely the distance something will diffuse comes out as an exponential based on time multiplied by a coefficient including size which, most proteins being made out of the same stuff, will corellate to mass.

 

So thickness of your splodge will be related to mass by an exponential

[math]W\propto e^m\times \text{Other stuff}[/math]

 

[math]\text{or }\\log(W)+C=m[/math]

 

Where m is mass, w is the width of the splodge and C is an arbitrary constant.

So you want to take the log of the band sizes and plot against mass of your known proteins

the paper will take the log for you, so just fit them on a scale of 0 to 10

ie. 100=10, 4=0.4

Then you can draw a line and use it to estimate the mass of the unknown.

 

This is a graphical method for linearizing your data and doing a linear regression (As DeltaScience was talking about). So it should achieve roughly the same result. However you will not have repeatable error bars etc. doing it graphically.

 

I could be entirely wrong on one of my assumptions, but given that you were provided with log paper mass will probably go on one axis and size on the other. If the dots are not forming a straight line at all then I probably have it the wrong way around.

Edited January 31, 2011 by Schrödinger's hat