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separated two protein


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#1 Poteo

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Posted 21 February 2005 - 12:56 AM

Hi, everybody
Now i can not separate two protein, which showed the closely Mw. Because showed one band on SDS-PAGE, but two band in Native. The native-PAGE, the two band were not so far mobility.

#2 Simonsays

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Posted 21 February 2005 - 12:10 PM

Hi proteo.
I'm not quite sure I understand what your problem is! From what I understand, you try to separate 2 proteins but they are too similar in mass. Is that it?
If so you could do a step of IEF (isoelectrical focusing) prior to SDS-PAGE. 2D-PAGE should get you there.

Simon

#3 sharath

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Posted 21 February 2005 - 08:26 PM

As your sample shows sinlge band on SDS PAGE and two on native PAGE, it indicates thatthey have similar mol.wt but slightly dissimmilar charge. Did you inlcude ion exchange chromatography in you protocol. Hydroxylapatite chromatography generallt resolves such close bands. Chramtofocussing can be used as separation is based on charge.
Sharath B.

#4 Poteo

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Posted 22 February 2005 - 08:41 PM

Thank sharath and Simon
Now i try to use Mono Q column for separating two protein, but my enzyme still bound to the column, even used 4 M NaCl in 50 mM Tris-HCl, pH 8.0 + 1 mM CaCl2. If you have some recommentation, please tell me.


Proteo

#5 sharath

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Posted 22 February 2005 - 08:54 PM

pH 8.0 is a bit high for ion exchange chromatography. try at 7.0 and may be even at 6.5 or 6.0

good luck

Sharath.B
National Chemical Laboratory,
India
Sharath B.




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