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SDS-Page Please help


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#1 silvepa23

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Posted 31 May 2011 - 09:13 PM

Hi,



I expressed a protein in E coli with a his tag. I was thinking about using an Ni Nta spin column which yields about 300 ug of protein. How does one check see how much protein is being expressed by the E coli? And how much protein should be added to a well on the SDS-page gel?

#2 protolder

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Posted 31 May 2011 - 09:47 PM

Hi,



I expressed a protein in E coli with a his tag. I was thinking about using an Ni Nta spin column which yields about 300 ug of protein. How does one check see how much protein is being expressed by the E coli? And how much protein should be added to a well on the SDS-page gel?

Hola, in a conventional PAGE of 8x10cm and 1.5mm thickness of 10 wells you could load about 50 ul in volume and 15-20ug of protein from a lysate. After run and stain you could calculate the proportion of your band respect the total protein of the well.More accurate is loading next to your lysate known amounts of a protein with similar mol. weight from 0.2to 2 ug. and compare and estimate how much protein you have in your extract. About amounts 10-20ug/well for extracts and 0.5-1-2ug for pure proteins. Buena suerte




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