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problem in cleavage of fusion protein
Posted by: isaackirubakaran (IP Hidden, New member, 7)
Date: September 2, 2005 02:28PM
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: isaackirubakaran@gmail.com Hope some will be able to help me out of this. Thanks Bye isaac
Re: problem in cleavage of fusion protein
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: September 2, 2005 03:40PM
It is possible that your cleavage was successful and the 9 kDA band is simply not being detected by your SDS-PAGE method. Small mw bands can be hard to detect. I have a few questions that might be of use in helping you. Are you detecting the protein in gel or transferring it to a membrane? If transferring the 9 kDa band may be going throught the membrane due to excessive current, transfer time or a large membrane pore size. Also if you are running the gel for too long a time, low molecular weight proteins can run off the gel. Use a loading dye or a standard marker with low molecular weights to keep track of the run and stop the run when the loading dye/low molecular weight marker reaches the bottom of the gel.
Re: problem in cleavage of fusion protein
Posted by: isaackirubakaran (IP Hidden, New member, 7)
Date: September 3, 2005 01:53AM
I am performing only SDS-PAGE after cleavage. After the complete run i stain the gel with CBB stain and i could not detect any band in the lower most. I have used both 12% and 15% gels for the same. I have not done western blot since i don't have antibodies for the protein. I will send your the expression gel photo as attachment. will you be able to tell that it is the fusion protein of size 40+9Kda. Please do mail me abt the same
Re: problem in cleavage of fusion protein
Posted by: isaackirubakaran (IP Hidden, New member, 7)
Date: September 3, 2005 01:54AM
I am performing only SDS-PAGE after cleavage. After the complete run i stain the gel with CBB stain and i could not detect any band in the lower most. I have used both 12% and 15% gels for the same. I have not done western blot since i don't have antibodies for the protein. I will send your the expression gel photo as attachment. will you be able to tell that it is the fusion protein of size 40+9Kda. Please do mail me abt the same
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