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Exctinction Co-efficient


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3 replies to this topic

#1 Prep!

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Posted 16 November 2009 - 01:44 AM

Hey all...
how do i theoratically determine the extinction co-efficient for a glycoprotein??!! is it even possible? the extinction will be of the native proteins and even sites like the expacy will give me extinction related to native protein without the PTM's but to find conc, i cant use this as even the glycans will contribute to the absorbance readings!! they do!!
Any suggestions?
Any easy approach for measuring it in the lab with limited resources??!!!
Any literature??
Thankx!!!
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#2 Feelcontraire

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Posted 18 November 2009 - 04:07 AM

Hey all...
how do i theoratically determine the extinction co-efficient for a glycoprotein??!! is it even possible? the extinction will be of the native proteins and even sites like the expacy will give me extinction related to native protein without the PTM's but to find conc, i cant use this as even the glycans will contribute to the absorbance readings!! they do!!
Any suggestions?
Any easy approach for measuring it in the lab with limited resources??!!!
Any literature??
Thankx!!!


You can cleave the glycan with PNGase, separate the protein and and glycan by any means (centrifugation,column, membrane...) and measure each absorbance separatedly.

If the PNGase digestion is mild and you perform an SDSPAGE you will observe two bands, one with glycan and another without. (You may get more than two bands if the PNGase finds more than one cutting place).

#3 Prep!

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Posted 18 November 2009 - 04:21 AM

Hey all...
how do i theoratically determine the extinction co-efficient for a glycoprotein??!! is it even possible? the extinction will be of the native proteins and even sites like the expacy will give me extinction related to native protein without the PTM's but to find conc, i cant use this as even the glycans will contribute to the absorbance readings!! they do!!
Any suggestions?
Any easy approach for measuring it in the lab with limited resources??!!!
Any literature??
Thankx!!!


You can cleave the glycan with PNGase, separate the protein and and glycan by any means (centrifugation,column, membrane...) and measure each absorbance separatedly.

If the PNGase digestion is mild and you perform an SDSPAGE you will observe two bands, one with glycan and another without. (You may get more than two bands if the PNGase finds more than one cutting place).


Thanx feel.. i am aware of al teh facts that you ve listed... i was intending to ask if tere was a method to determione the extnction without cleaving the glycans. IT is not feasable to cleave the glycan each time to measure protein conc as the efficiency ,ay also nit be same and the further analysis will be based on the glycosylated molecule!!!
thanx anyways
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#4 Feelcontraire

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Posted 18 November 2009 - 04:30 AM

Hey all...
how do i theoratically determine the extinction co-efficient for a glycoprotein??!! is it even possible? the extinction will be of the native proteins and even sites like the expacy will give me extinction related to native protein without the PTM's but to find conc, i cant use this as even the glycans will contribute to the absorbance readings!! they do!!
Any suggestions?
Any easy approach for measuring it in the lab with limited resources??!!!
Any literature??
Thankx!!!


You can cleave the glycan with PNGase, separate the protein and and glycan by any means (centrifugation,column, membrane...) and measure each absorbance separatedly.

If the PNGase digestion is mild and you perform an SDSPAGE you will observe two bands, one with glycan and another without. (You may get more than two bands if the PNGase finds more than one cutting place).


Thanx feel.. i am aware of al teh facts that you ve listed... i was intending to ask if tere was a method to determione the extnction without cleaving the glycans. IT is not feasable to cleave the glycan each time to measure protein conc as the efficiency ,ay also nit be same and the further analysis will be based on the glycosylated molecule!!!
thanx anyways


Well, I was thinking more on the side of cleaving the glycan and measure it's absorbance and integrate it with the glycan-less protein absorbance. So you would probe it's your protein and would know the absorbance you have to check the next time you search for your glycoprotein. Thus you only have to cleave it the first time.




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