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cloning into pEGFP-C1 vector

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cloning into pEGFP-C1 vector

Postby baaje » Sun Apr 25, 2010 8:08 am

hi all,
i am very new to cloning work. i have cloned my gene into pEGFP-C1 vector (my gene would be in the C-terminus of GFP). it was confirmed by sequencing! my problem is when i express in cells(i can see GFP signal) but when i do western blot..i can see only GFP signal (~30 kDa) not GFP+gene signal! which is appro. around 75 kDa.
this vector already has start codon(that's why i can see GFP signal) and i have also included start codon in my gene. do you... guys think i shouldn't ve put start codon in gene? i am confused....i am totally new to this....
help me
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Postby JackBean » Sun Apr 25, 2010 8:21 am

the start codon shouldn't matter (it's read as Met;), but did you check the stop codons? Is your gene in frame with GFP?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: cloning into pEGFP-C1 vector

Postby baaje » Sun Apr 25, 2010 9:34 am

thank u for ur reply,
yeah! i've checked...my insert is in frame...even its not in frame...still it should be expressed as GFP-my gene, i could see GFP signal when i transfect cells but could see only GFP band around 30 kDa (like GFP alone transfected cells) not at 75 kDa(GFP-my gene)
Yeah..i checked stop codon..its ok
its totally confusing!!
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Postby JackBean » Sun Apr 25, 2010 11:37 am

it must be in frame to be it expressed as fusion protein :roll: Isn't there some site for protease cleavage?
http://www.biolib.cz/en/main/

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Re: cloning into pEGFP-C1 vector

Postby baaje » Tue Apr 27, 2010 1:50 am

hi
as u said i should see...any protease can cleave...
I have one more question about "in frame"..see...my vector is start codon-GFP-MCS-Stop codon.
i put my gene in MCS.....even if its not in frame...still it shoud express GFP-off frame protein, so.. definitely i should see shift (apprrox. 75 kDa) in SDS-PAGE gel if i run the sample after expressing in the cell (or am i wrong??)...but i just see only 30 kDa band which is exactly like control GFP alone.
my question is ...is it possible that only GFP can express, if my protein is not in frame??
thank you for ur time.
bye
baaje
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Postby JackBean » Tue Apr 27, 2010 7:41 am

Definitelly. You are probably not much able to distinguish between 30 and 32 kDa ;) And the translation machinary takes the first STOP codon, which it reaches, so if in your reading frame is some premature STOP codon, the protein will be definitely interrupted ;)
http://www.biolib.cz/en/main/

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Re: cloning into pEGFP-C1 vector

Postby baaje » Tue Apr 27, 2010 12:42 pm

hi...i got it!! :oops:
Why didn't i think... the way you think?! :oops:
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