11 replies to this topic

[proteinrunner]

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

Edited by proteinrunner, 20 May 2010 - 01:02 AM.

[Inmost sun]

proteinrunner, on May 20 2010, 09:01 AM, said:

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

coexpression with GFP or an analogue to build a fusion protein...

[ProteinWork]

Inmost sun, on May 20 2010, 06:53 PM, said:

proteinrunner, on May 20 2010, 09:01 AM, said:

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

coexpression with GFP or an analogue to build a fusion protein...

GFP alone is ~27kDa, almost 3 times as the size of proteinrunner's peptide. I think that might cause some problems with the peptide's correct folding and localization.

[swanny]

ProteinWork, on May 21 2010, 09:18 AM, said:

Inmost sun, on May 20 2010, 06:53 PM, said:

proteinrunner, on May 20 2010, 09:01 AM, said:

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

coexpression with GFP or an analogue to build a fusion protein...

GFP alone is ~27kDa, almost 3 times as the size of proteinrunner's peptide. I think that might cause some problems with the peptide's correct folding and localization.

Not necessarily. GFP is known to increase stability, and certainly solubility of fusion partners.

Where do you predict the protein should be (I think we can call a 10 kDa polypeptide a protein...)?

[ProteinWork]

swanny, on May 21 2010, 12:52 AM, said:

ProteinWork, on May 21 2010, 09:18 AM, said:

Inmost sun, on May 20 2010, 06:53 PM, said:

proteinrunner, on May 20 2010, 09:01 AM, said:

Hi,

I'm transfecting cells with a construct producing a -10kDa peptide which is very easily degraded. Is there any technique that I can use to visualize the peptide's localization in the cell? I've tried the normal staining but I can't see any thing. I can't even see the WB signal of it.

I hope somebody can answer the question...

Thanks,
Proteinrunner

coexpression with GFP or an analogue to build a fusion protein...

GFP alone is ~27kDa, almost 3 times as the size of proteinrunner's peptide. I think that might cause some problems with the peptide's correct folding and localization.

Not necessarily. GFP is known to increase stability, and certainly solubility of fusion partners.

Where do you predict the protein should be (I think we can call a 10 kDa polypeptide a protein...)?

I think proteinrunner was trying to determine the localization pattern of this protein. I'm just not sure how GFP fusion will affect it. But I don't know any other way to do it, either.

[proteinrunner]

Thank you guys!!

I expect it to be a nuclear protien as a transcription factor. The construct of it has a myc tag, but it's not useful either for WB or IF... We have another longer pepteide (kind of precurcor of the 10kDa peptide) with gfp tag, but we can't see it by IF either...

Any other ideas??

Have a nice day!

[bob1]

If it is myc tagged, you should be able to detect it by antibody - there are lots of Myc antibodies about.

[proteinrunner]

I\ve already tried that, and it didn\t work...

bob1, on May 23 2010, 05:03 PM, said:

If it is myc tagged, you should be able to detect it by antibody - there are lots of Myc antibodies about.

[laurequillo]

proteinrunner, on May 26 2010, 09:36 AM, said:

I\ve already tried that, and it didn\t work...

bob1, on May 23 2010, 05:03 PM, said:

If it is myc tagged, you should be able to detect it by antibody - there are lots of Myc antibodies about.

Maybe you could try to increase the stability of the protein. Using a proteosome inhibitor or something like that.

[medchemgirl]

As I understand, u are just expressing a small peptide, so it's not a protein perse, is it just a fragment of a protein? Why don't you express the whole thing tagged to GFP? you should definitely see the GFP construct. Make sure you are not fusing the antisense, otherwise you won't get it, and it has to be in frame. Check your sequence.

[proteinrunner]

We haven't done that yet. We have a precursor of this peptide, a longer fragment from the holoprotein, with gfp tagged. But I can't see anything green by overexpressing this tagged protein fragment. I'm afraid the gfp tag won't work for my interested peptide as well.

medchemgirl, on May 26 2010, 07:22 AM, said:

As I understand, u are just expressing a small peptide, so it's not a protein perse, is it just a fragment of a protein? Why don't you express the whole thing tagged to GFP? you should definitely see the GFP construct. Make sure you are not fusing the antisense, otherwise you won't get it, and it has to be in frame. Check your sequence.

Edited by proteinrunner, 28 May 2010 - 03:17 AM.

[rkay447]

First, have you done the control transfection of the GFP only so that you know your transfection, cells and DNA are good?  If you can see the control GFP but not your GFP-tagged peptide, then something is wrong with the DNA.  Is your peptide tagged on the NT or CT?  If it's the CT, I'd be concerned that the GFP is not in the correct frame or that you've got a mutation that created a stop codon early on in the GFP.  Also, make sure you have a good Kozak sequence.  Recheck the sequence carefully!!  The other idea I have is that for some reason that I can not explain, I have one construct in particular that the transfection or expression (not sure which) declines rapidly with each and every freeze/thaw.  It is the only construct that has this particularity and after about two freeze thaws, it's completely dead.  Just try to do a quick miniprep and make a fresh DNA prep before the transfection.