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Western Blot Protein Length

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Western Blot Protein Length

Postby rayman » Tue Aug 30, 2011 12:22 pm

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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Postby JackBean » Tue Aug 30, 2011 12:49 pm

the mobility (expressed as distance of band from the start) is proportional to log M_r
So, if you want to have it precise, measure distances from start to respective bands, calculate log of their molecular weight and put into graph. By calculating the equation, you can calculate M_r of your proteins
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Western Blot Protein Length

Postby rayman » Tue Aug 30, 2011 7:32 pm

Thank you for your answer.

I have some questions left.

What do you mean by M_r ?

How can I put them into the graph? What is the equation?
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Postby JackBean » Tue Aug 30, 2011 8:04 pm

M_r is the molecular mass (or relative weight, that's the same, except units)

on x-axis you put log M_r; on y-axis you put the distance and let the Excel calculate the equation
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Western Blot Protein Length

Postby rayman » Tue Aug 30, 2011 8:13 pm

Thank you for your answer.

I need help with Excel.

Can you tell me what I have to type in?

Do you use any kind of messengers so I can ask you in a faster way?
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Re: Western Blot Protein Length

Postby rayman » Tue Aug 30, 2011 8:37 pm

Why do I have to use the log of MW ?
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Postby JackBean » Tue Aug 30, 2011 10:16 pm

because otherwise you would have logaritmic line, what is not that big problem today, since we use computers anyway, but using log is kind of more convenient ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Western Blot Protein Length

Postby rayman » Wed Aug 31, 2011 12:18 pm

Thank you.

I read on other websites that there is another way to solve this.

They basically follow the same procedure but instead of only measuring the distance from the starting point they divide the length of the protein by the distance from the starting point.

So you have another coordinate system with the log(MW) on the x-axis and the division of distance over length on the y-axis.

Which way do you recommend? Yours or the above mentioned?
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Postby JackBean » Wed Aug 31, 2011 8:56 pm

what is length of protein? Like 35 kDa/5 cm?
http://www.biolib.cz/en/main/

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Postby merv » Mon Oct 03, 2011 2:07 am

i suppose teh corner shop was fresh out of common sense...
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