Cleavage of Maltose-binding fusion protein

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isaackirubakaran
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Cleavage of Maltose-binding fusion protein

Post by isaackirubakaran » Fri Sep 02, 2005 7:10 pm

problem in cleavage of fusion protein (MBP-fusion protein)

I am having some problem in my work related to cleavage of maltose-binding protein from the protein of my interest. I have cloned a plant LTP gene into pMAL vector and now i have got the expression of 49 kDa fusion protein (40kDa MBP + 9 kDa LTP). when i cleave the fusion protein with the enzyme Factor Xa (Protease) then run SDS-PAGE i could not detect the bands of 9 kDa protein but i could detect the MBP alone. What would be the reasons if so what can be done to detect the protein of interest 9kDa LTP.

My conditions for Factor Xa cleavage :

1 micro litrel of enzyme was used to cleave 50 microgram of protein (fusion protein).

Incubation at 25 *C for 6 hours, 8 Hours and 12 Hours.

All the sample showed similar type of results. (No band of the low mol. Wt. Protein 9KDa.

What are the methods that can be adopted for the detection of the cleaved protein. Whether native PAGE can be done or any other method will be efficient and useful.

Please do help me. mail me you valuable suggestions and ideas to

my email id: [email protected]

Hope some will be able to help me out of this.

Thanks Bye

isaac

sdekivit
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Post by sdekivit » Fri Sep 02, 2005 7:38 pm

most p[recise method for detecting proteins is MS (MALDI-TOF)

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