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His-tag fusion protein expression trouble, help me

His-tag fusion protein expression trouble, help me - Protocols and Methods Forum

His-tag fusion protein expression trouble, help me - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 02-17-2004, 09:00 AM
YingFang_Yang@itri.org.tw
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Default His-tag fusion protein expression trouble, help me



I cloned a 43kD and 40kD genes into invitrogen's pTrcHis vector. The
direction and sequese are all correct. But I get troubles in expressing the
two gene protein products. There are major bands at right place on SDS-PAGE
after Ni-NTA purification but the major bands can not be detected by
anti-His Ab and western bloting. There is a band dtected arond 30kD by
western bloting. The results are too wired to me.

Could anyone give me some suggestion?

Any suggestion is appreciated.

Best


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Old 02-17-2004, 03:44 PM
EK
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Default His-tag fusion protein expression trouble, help me


<[Only registered users see links. ].tw> wrote in message
news:[Only registered users see links. ].tw...
the
SDS-PAGE
1. One possibility is that you anti 6His Ab is bad. Check with a positive
control.
2. The lower MW might be the result of early termination of transcription.
Check if you cloned inserts have alternative stop codons inside the
sequence.
3. Check if there are known sites recognized by E.coli proteases in the
seauences of your 2 proteins. It is possible that you get efficiently
cleaved product missing 6His.


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