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Problem with GFP detection in western

gfp western

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#1 Rrad5

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Posted 13 June 2013 - 04:36 AM

Hi,
I'm trying to trouble shoot a western blot. We're currently using a protein that's tagged with myc, and have that western running just fine. But it would be better if we could take an aliquot of sample that's GFP tagged (yeast strain) that we're imaging and directly use it in our westerns as well.

The problem is that we can't seem to detect the gfp tagged protein in our westerns. I've tried using a Roche anit-GFP, Cat. No. 11 814 460 001. Someone else in the lab has used the same antibody with no problem. I'm using the same conditions (1/1000, blocking with 2% milk) and get nothing.

Any suggestions would be great.

Thanks

#2 mdfenko

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Posted 13 June 2013 - 04:51 AM

are you using the same protein as your labmate?

if not, have you checked the transfer efficiency of your gfp-protein?
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#3 Iceage

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Posted 13 June 2013 - 05:46 AM

Yes, first things I would test are the efficiency of transfer and quality of lysates/protein extracts. you could probably just stain the gel to test is the proteins are getting degraded. I am assuming you do not have an Ab for your protein of interest? Thats ofcourse would be the best way to test. Another thing you could try is to immunoprecipitate the fusion protein and then run on a gel. Do you know if the protein forms multimers? You do mention that myc tag worked fine. Did you try running on a gradient gel? You could also try using more Ab. it seemed to work for some of my blots (1:500 or more) .The last option would be to change the Ab. I have used living colors living peptide ab with no problems. I never used the Roche Ab. Good luck!

#4 Rrad5

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Posted 14 June 2013 - 01:53 AM

are you using the same protein as your labmate?

if not, have you checked the transfer efficiency of your gfp-protein?

I'm using whole cell lysates (TCA extraction or RIPA buffer, neither work), so I don't know how I'd test for the transfer efficiency. IPs are tricky even with the myc tag.

#5 Rrad5

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Posted 14 June 2013 - 01:55 AM

Yes, first things I would test are the efficiency of transfer and quality of lysates/protein extracts. you could probably just stain the gel to test is the proteins are getting degraded. I am assuming you do not have an Ab for your protein of interest? Thats ofcourse would be the best way to test. Another thing you could try is to immunoprecipitate the fusion protein and then run on a gel. Do you know if the protein forms multimers? You do mention that myc tag worked fine. Did you try running on a gradient gel? You could also try using more Ab. it seemed to work for some of my blots (1:500 or more) .The last option would be to change the Ab. I have used living colors living peptide ab with no problems. I never used the Roche Ab. Good luck!

No Ab for the protein. If we made it for everything we're looking at it would get hella expensive.
IP is tricky with the myc tag as it is. I'll try the gradient gel and increased Ab. Looking around, it seems very few people use the Roche. Will try a different brand. Thanks for your suggestions.

#6 mdfenko

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Posted 14 June 2013 - 04:48 AM

are you doing western blotting or immunoprecipitation (ip)?

our responses are based on western blotting.

you determine transfer efficiency by staining the gel after transfer to see if and what is left behind. you can also reversibly stain the membrane (before blocking) with ponceau s but it isn't as sensitive.
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#7 labtastic

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Posted 07 July 2013 - 04:20 PM

If you tag your protein with GFP and only want to detect GFP-tagged protein, remember that GFP is stable in SDS (so long as you don't boil you samples prior to PAGE). Run your SDS-PAGE gels as usual and then image your gel directly with a fluorescence imager . No blotting necessary, sensitivity rivals that of blotting. And it takes 2 seconds rather than all day. Just an idea... Posted Image

Edited by labtastic, 07 July 2013 - 04:20 PM.






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