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IF Two Antigen Retrival Steps


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#1 DavidEckert

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Posted 31 January 2017 - 09:03 AM

I have an interesting scenario. I'm focused on the colocalization of two proteins via IF. Protein #1 performs much better with the 20 minute pH 6.0 citrate buffer antigen retrival method, protein #2 performs much better with the 20 minute Tris-EDTA pH 9.0 method. Is it possible to do two antigen retrival methods, one after the other? Has anyone done this?



#2 bob1

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Posted 31 January 2017 - 09:12 AM

I haven't done it, but I would be concerned that you could damage the first antigen by performing the second recovery. However, both methods you are using there are relatively mild, so I would guess it will work. I guess your only option is to try it and see.



#3 DavidEckert

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Posted 07 February 2017 - 09:48 AM

I gave it a shot, I did the pH 9.0 20 min boiling first followed by the 20 min boiling in pH 6.0.

 

5% of the sections look amazing, the other 95% have no signal. Very inconsistent staining. Do you think it has to do with uneven heating in a domestic microwave or was too much protein degraded?



#4 mdfenko

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Posted 08 February 2017 - 04:27 AM

can you try a more uniform heating method (eg hot plate)?


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