His-tag protein is lost - (Jul/19/2006 )

I used pET28 to overexpressed his-tag protein, verifed protein by Western-blotting (several times) and proceeds with purification. But I can not get the protein after 2 months, all the conditions are the same, last week I re-transformed plasmid into expressed host but no protein appeared. I am using BL21(BE3).

Is there anyone who has this kind of experience before? thanks in advance for any kind advices.

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i also expressed my gene product but in pET 23D and then transformed BL21 cells. ur main problem is in purification so if u are using Ni-NTA affinity columns then check for the concentration of imidazole buffers.it may be because the nickel beads are not regenerated properly.

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hi
tell me onething when u r doing western blotting r u checkingby loading the following , cell lysate and host alone(host not transformed plasmid ) if yes in that u r getting any band means and unable to get after purification means that some thing problem with u r down stream, if u unable to get the band in the lysate itself means that upstream process is a problem just check the expression levels by inoculating the from glycerol stock and tell me in detail where exactly u r
bye

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1 BL21发生了突变
2 IPTG是否失效?

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Maybe you should check your Bl21, this once happened to me and it turned out that the comp cells were rather old and when i made them fresh the protein expressed fine.

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Thanks for your kind advices. I have expressed the protein again, it seems the plasmid were lost or something went wrong with IPTG, cause the protein came out after I re-transform the plasmid into host and change another brand of IPTG.

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