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Protein expression using invitrogen pSecTag2


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#1 SDP

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Posted 16 December 2009 - 07:47 AM

Hi everyone,
I have been working with Invitrogen's mammalian expression vector pSecTag2. This vector consists of a immunoglobulin signal peptide which enables extracellular secretion of the protein being expressed by the transfected cells. I have cloned 4 different constructs (essentially the same genomic sequence with 2 mutations and hence 4 haplotypes) in frame with the start codon on the 5 prime end and the myc and his tags at the 3 prime end. Currently I am doing transient transfections (I harvest my supernatant 72 hours post transfection) in a 6 well plate (4 ml of culture medium) as I require just enough protein to do an ELISA later on. However, I have failed to detect any protein on a western blot when using anti his tag-HRP antibodies. I am wondering if anyone has any experience doing transient transfections, especially with the pSecTag2 vector (consists of a CMV promoter) and if anyone can give me an idea if 4 ml of supernatant could give yield sufficient protein (lets say 1 microgram) for an ELISA.


Thanks to you all

Cheers

Sam

#2 Benita

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Posted 07 January 2010 - 01:12 AM

Hey Sam,

I'm working with this vector, too. For me it works quite well. Maybe you can try to check your method by doing a transfection with pSectag2-PSA (delivered by invitrogen as a control). For that you should see a high signal by using ant-His-HRP antibody.

Do you use the supernatant for your western? So I always made a Nickel-Bead purification before the western blot. Maybe thats the problem?

Do you have FCS in your media in what your protein is secreted?

Greetings,

Benita!

#3 SDP

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Posted 07 January 2010 - 06:48 AM

Hey Sam,

I'm working with this vector, too. For me it works quite well. Maybe you can try to check your method by doing a transfection with pSectag2-PSA (delivered by invitrogen as a control). For that you should see a high signal by using ant-His-HRP antibody.

Do you use the supernatant for your western? So I always made a Nickel-Bead purification before the western blot. Maybe thats the problem?

Do you have FCS in your media in what your protein is secreted?

Greetings,

Benita!


Hey Benita

Thanks for the reply. I will try the transfection with pSecTag2-PSA and see if i can get that working. I am using anti-His-HRP from abcam and use the supernatant to check for protein expression. Initially i was using anti myc antibody coated beads for purification. But after purification and running a SDS PAGE / Western.. i do not see anything. I dont know what FCS is.. if you mean FBS (fetal bovine serum), then yeah.. i do have that in my media. But during the 6 hours of transfection i use optimem alone which does not contain FBS.

On a different note, could you perhaps comment on what ratio of the vector to lipofectamine do you use? (if you use lipofectamine i.e.) and what culture vessel do you use .. e.g. a t75 flask or 6 well plate? I have been using 6 well plates with about 4 ml of culture supernatant. I require 2 - 3 micrograms of expressed recombinant protein. Do you think that a 6 well plate could give me that much?

Thanks a bunch for your reply.

Regards

Sam




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