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Topic: Thawing Frozen Serum Samples for ELISA  (Read 3571 times)

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Offline lab2015

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Thawing Frozen Serum Samples for ELISA
« on: November 29, 2016, 09:44:08 AM »
I will do ELISA to detect a protien in serum samples. Samples are stored in -80. I will thaw samples on ice. Do I need to vortex or spin these samples before adding to the microplate?

I would appreciate your advice.

Many thanks
C

Offline lab2015

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ELISA Protocol
« Reply #1 on: November 29, 2016, 09:56:27 AM »
I have a question regarding the protocol of ELISA kit I am using.
It's a sandwich ELISA. After adding samples and standards, the protocol asks to "remove the liquid of each well, don't wash"
how should I remove the liquid? should I use the pipette? should I snap the plate onto absorbant paper?
the next step is to add detection reagent and then to wash. would the expermint be affected if there was some liquid left from the previous step?

I would appreciate your help.

Many thanks,
C

Offline Yggdrasil

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Re: Thawing Frozen Serum Samples for ELISA
« Reply #2 on: November 30, 2016, 11:50:11 AM »
I will do ELISA to detect a protien in serum samples. Samples are stored in -80. I will thaw samples on ice. Do I need to vortex or spin these samples before adding to the microplate?

Because many antibodies used in ELISA assays recognize only correctly folded proteins, it will be important to avoid steps that may denature your protein sample.  Thawing samples on ice in general is a good idea to help avoid denaturing your protein.  Mixing should be done gently (i.e. invert the tube a few times or gently tap the side of the tube).  You should definitely avoid vortexing.  Centrifugation could help if you have a lot of precipitate in the tube (but make sure that your protein of interest is not in the precipitate before you throw it out!).

Quote
It's a sandwich ELISA. After adding samples and standards, the protocol asks to "remove the liquid of each well, don't wash"
how should I remove the liquid? should I use the pipette? should I snap the plate onto absorbant paper?
the next step is to add detection reagent and then to wash. would the expermint be affected if there was some liquid left from the previous step?

It's hard to provide guidance without knowing the details of the protocol, but if the protocol calls for removing the liquid without washing, then residual liquid from the previous step is unlikely to interfere with the next step.  Pipetting, aspiration, or snapping the plate would all be acceptable methods of removing the liquid (as long as the supernatant does not need to be saved, at least).


Offline lab2015

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Re: Thawing Frozen Serum Samples for ELISA
« Reply #3 on: November 30, 2016, 12:32:14 PM »
Thank you so much for your help.

C

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