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Forgot to block after stripping - please send help

stripping; western blot

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#1 NeuroG2019

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Posted 15 November 2017 - 08:18 AM

Well, my boss is of course livid.

 

This Western has been an ongoing problem child, but I feel that the cherry on top was last night, when I finished stripping the membrane at 10:30 then decided to start the primary after all.

 

But, I was so occupied with which protein I should reprobe for, that I completely forgot you need to block after stripping. Which, frankly, had I remembered: I would not have started the primary that night.

 

Here's my standard procedure:

 

Strip with 2.5pH glycine, 0.2M for thirty minutes

Wash three times in PBS-T, 10 minutes each

Image to test for residual antibody

Block in 3% BSA in PBST, 1 hour room temp

Incubate in primary overnight

 

Except I forgot to block. So it went straight into primary antibody after washing. Does anybody know how bad that is, on a scale of 1-10? My boss doesn't, because he apparently doesn't make mistakes, ever. I believe his exact words were "I don't know, because I've never done that." (Followed by "'It was late' is not an excuse!" For a neuroscientist, he certainly doesn't seem to have much appreciation for the effects of exhaustion or anxiety on student function.)

 

This membrane has already been stripped twice. I'm loath to do it a third time, but not sure I have any choice.

 

It's not like I wanted to forget...unsure.png



#2 mdfenko

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Posted 16 November 2017 - 02:43 PM

complete the blot. stripping with glycine alone should only remove the antibodies from the transferred proteins. the proteins and the blocking agent should remain bound to the membrane. reblocking, under those conditions, should not be necessary.


talent does what it can
genius does what it must
i do what i get paid to do




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