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ELISA positive result from Phosphatase/Protease inhibitor?

ELISA Protease false positive

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3 replies to this topic

#1 Rnotk

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Posted 15 February 2018 - 04:21 PM

Hi All

 

I heard something interesting about ELISA.

One person was doing ELISA to quantify phosphorylated protein, got abnormally large signal.

Further investigation showed the signal was coming from protease/phosphatase inhibitor not from the sample or matrix effect.

 

Have you experience the similar issue before?

It is a bit difficult for me to believe and I cannot even find any publication stating that. But I am very curious.

 

If you happen to know any info, scientific explanation or publication demonstrating that, please let me know!!

 

Thank you in Advance 



#2 bob1

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Posted 16 February 2018 - 06:37 AM

Not quite the same, but many blocking proteins are phosphorylated, especially if they are milk derived. So you should avoid using milk derived blocks  if you are looking at phosphorylated proteins.



#3 Rnotk

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Posted 16 February 2018 - 09:37 AM

Hey Bob

 

Thank you for the reply!

 

Yes, he is quantifying phosphorylated protein, and I think the sample was mouse serum or plasma.

The ELISA kit he used was commercially available, although I do not know what blocking was used. (But I think it was BSA base or casein base).

 

I also suspect he might be using PBS, but this was not the case.

 

So I am interested in what is the science behind this.



#4 bob1

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Posted 16 February 2018 - 10:04 AM

Casein is a milk protein and is definitely phosphorylated. Avoid BLOTTO and casein based buffers in general for this sort of work, though I have heard of them working with particular antibodies. BSA should be OK. You could also try polyvinylpyrrolidone (PVP) blocking.







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