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transgenics and controls show same levels of expression!!


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#1 scary

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Posted 03 April 2009 - 08:24 PM

Hi,

Plz some one help me. I am working with a set of transgenic crops like Pigeon pea, Field bean, chickpea, groundnut and cotton which have nptII as a marker gene. I have raised the antibodies for nptII and found it working very well for western blots. My problem is when ever I carryout an ELISA for the same samples the results will be horrible. My controls and transgenics show almost the same values when my buffer blanks are without any background.

My boss is trashing me very badly as I am not able to sort out the problem. Plz some one help me.

Thank U,

#2 Carlton H

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Posted 05 April 2009 - 04:13 PM

Is your antibody enzyme-linked or are you detecting with fluorescence using a secondary antibody? If so, has anyone ever used that secondary for fluorescence ELISA or other fluorescence methods such as IHC? If not, it may indeed be a problem with your antibody as there are many antibodies that work in western that do not work well for other fluorescence-based methods.

-Carlton

Edited by Carlton H, 05 April 2009 - 04:13 PM.

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#3 scary

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Posted 05 April 2009 - 09:35 PM

Is your antibody enzyme-linked or are you detecting with fluorescence using a secondary antibody? If so, has anyone ever used that secondary for fluorescence ELISA or other fluorescence methods such as IHC? If not, it may indeed be a problem with your antibody as there are many antibodies that work in western that do not work well for other fluorescence-based methods.

-Carlton



Hi Carlton,

Thanks for the reply. I am using a secondary antibody conjugated with enzyme which I will later read with a colorimetry based ELISA reader. As you said the problem might be with my antiboy it self but people are using the same antibody to select the transgenic crops throughout the world so I do not know what to conclude?

Thank U
-Scary

#4 Carlton H

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Posted 10 April 2009 - 07:58 AM

What controls are you doing? Are you sure that your primary is binding to the protein and / or that your primary is binding to your secondary?

A few ideas to troubleshoot:

Try coupling purified nptII protein to a sepharose bead, or something similar, then go through your ELISA protocol and see if you can see the protein. Use multiple secondaries (I'd recommend at least 3). If you can see fluorescence with other 2* Abs, but not the one you've been using, then the 2* is definitely the problem. If you don't get staining in ANY of them, then it's more likely that your 1* just isn't working (or at least isn't suitable for your ELISA protocol).

-Carlton

P.S. - If you do any field work, you should check out our bullet blender. It's the only homogenizer really suitable for use in the field so you can prep samples wherever you are with no cross-contamination. http://www.nextadvance.com/index.htm Thanks!
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#5 scary

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Posted 10 April 2009 - 11:16 PM

Hi,

Thank u. Coming to the troubleshooting ideas that you have suggested, I have already tried the first part of it. i.e., purification of serum against the nptII protein in a sepharose column. The outcome was same and my results didnot make any difference!!
The second part, trying different 2* I will try that and hope it will work.

Thanks a lot.
-Scary

#6 Carlton H

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Posted 13 April 2009 - 05:49 PM

If I can be of any further help, just let me know!
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