Western Blot

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Bio2013
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Western Blot

Post by Bio2013 » Wed Jan 30, 2013 7:00 am

Hi BIologists,
I have a question, how can we know about the size of protein in the western blot? I know the full lenght, but after WB, I am seeing band with a different size!
Thanks for your help.

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JackBean
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Post by JackBean » Sat Feb 02, 2013 11:17 pm

What about post-transcrioptional modifications?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Bio2013
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Re:

Post by Bio2013 » Mon Feb 11, 2013 5:26 am

JackBean wrote:What about post-transcrioptional modifications?

Actually, I do not know exactly how to measure post translational modifications and reduce them from the full size of the protein, can any body help me with that? would post translational modifications be same for all proteins, and how can I find out about that?
thanks

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JackBean
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Post by JackBean » Mon Feb 11, 2013 3:31 pm

Those affecting size on SDS-PAGE would be mostly glycosylation or proteolysis, depending on the size of your band. Or the mobility of your protein is affected for some reason. Could you measure size of your protein on MALDI MS?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

Bio2013
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Re:

Post by Bio2013 » Sat Feb 16, 2013 8:48 pm

JackBean wrote:Those affecting size on SDS-PAGE would be mostly glycosylation or proteolysis, depending on the size of your band. Or the mobility of your protein is affected for some reason. Could you measure size of your protein on MALDI MS?


sorry, but I do not know what MALSI MS is.

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JackBean
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Post by JackBean » Sat Feb 16, 2013 9:57 pm

mass spectrophotometer
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Post by MarkHolland » Sat Mar 30, 2013 3:22 pm

Separating Proteins by Size

To begin characterizing a protein, it is often useful to be able to separate it from as many other proteins in the mixture as possible. One of the first techniques that can be used is to separate it according to how big it is, otherwise known as its molecular weight. When extracts are prepared from a cell, detergents can be used to make the proteins unfold into the linear chain of amino acids. Using a detergent such as sodium dodecyl sulfate (SDS) makes proteins unfold and gives them a net negative charge that is directly related to their molecular size. Using this feature, Dr. U. K. Laemmli developed a system of gel electrophoresis through a plastic medium (polyacrylamide) that easily allowed for proteins to be separated by an electric current strictly according to their overall mass. (1)

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