special band in BL21(DE3) - protein expression (Apr/18/2006 )

friend to why i can not expression the protein?
vector(pET32a including my gene) tranfer to BL21(DE3) to expression my protein ,but in SDS-PAGE, i could not find any band of 43 kd.26 kd of my protein and 17 kd of tag proteia. instead i am getting band round about 25 kd.why ?
thanks

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could be many things

would you please give more details about your induction protocol?

a possibility is degradation or post-translational modification that has resulted in truncated or degraded protein

another possibility is that the clone is not what you think it is...have you sequenced it?

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chances are there that ur desired fragment is not in frame so, u chek it first and also
B) try it in some other media , i don't know which one u used first but also try YT and TB.
C) Try some other avialable T7 expression host like C-41(DE3) (Novagen).
D) induce ur culture with IPTG when O. D. will reach around 1 or 1.2.
All the best

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To aimikins:
1.culture 37℃ and when O. D. will reach around 0.6 or .08 add IPTG(1mM) to induce.
2.collect after 1h ,2h,3h ,4h.
3.in 4℃ 4000rps 10min discard water and add 100 ul ddH2O,100 ul 2XBuffer.20 ul β-Mercaptoethanol .boiling 10min then SDS-PAGE

To sarvind:
i am using LB culture ,thanks for your advice ,i will try other culture.

thanks aimikins and sarvind advice

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Hi ily,

I also have the same problem when I am trying to express a self designed gene construct , using pET19b expression vector in either Bl21(DE3) or Rosetta(DE3). I also see a SDS Page band corresponding to ~25Kd. I tried both LB and 2xYT media, but results are same.

Could you by any chance come up with any idea against this problem ?

Suggestions from all are most welcome.

thanks,
odk

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can u try ti induce IPTG at 1.2or 1.6 , i read that some proteins cant be expressed in 0.6 OD.
another question: ur BL21 bacterial plate.. how long it has been since u transformed it and make a plate?
once i didnt get my expressed protein because my plates were 2 weeks old .

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Please read this link its great

http://www.embl.de/ExternalInfo/protein_un...exp-system.html

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For some proteins I am trying to purify, I have a strong band at 70 kDa........ wish could correspond to one of the bacteria chaperone (DNAK)

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You might want to check out the plasmid vendor's website (I think it's novogen?). I have a feeling that your mystery band might be the T7 pol.

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