Dec 01, 2023

Direct ELISA

DOI
https://dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
  • Michael X. Henderson1
  • 1Van Andel Institute
Michael Henderson
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DOI: https://dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
Protocol Citation: Michael X. Henderson 2023. Direct ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged83ov47/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 29, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 91706
Keywords: ASAPCRN, direct elisa this protocol, direct elisa, protocol detail, protocol
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020616
Abstract
This protocol details Direct ELISA.
Attachments
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911-2362.pdf
169KB
Direct ELISA
13h 33m
Dilute 100 ng - 500 ng of protein of interest (e.g. α-synuclein) in Takeda buffer per well and add 30 µL total liquid per well to 384-well Nunc Maxisorp plate.

Seal with removable clear adhesive cover.
Centrifuge the plate at 1000 x g for 00:01:00 to pull down protein onto the plate. Leave for 04:00:00 at 37 °C or Overnight at 4 °C .

4h 1m
Use the plate washer with 5x with 100 µL PBST.
Block with 100 µL Blockace per well. Fill wells from the bottom, being sure to avoid leaving any bubbles in the wells.

Seal with a removable clear adhesive cover and leave for 04:00:00 at 37 °C orOvernight at 4 °C .

Note
At this point, plates can be stored for up to 1 month at 4 °C if there is a preservative in the buffer.





4h
Use the plate washer with 5x with 100 µL PBST.

Use C buffer to dilute reporter antibody. Vortex immediately before pipetting.
Using multichannel, fill 91, dispense 30 µL three times.

Seal with removable clear adhesive cover and centrifuge plate at 1000 x g for 00:01:00 .

1m
Incubate for 04:00:00 at 37 °C or Overnight at 4 °C .

4h
Use C buffer to dilute HRP-conjugated secondary reporter antibody.
Add 30 µL per well. For goat-anti-mouse/rabbit use at 1:5-20K.

Seal with removable clear adhesive cover and centrifuge plate at 1000 x g for 00:01:00 .

1m
Incubate for 01:00:00 at 37 °C .

1h
Use the plate washer with 5x with 100 µL PBST.

Add 30 µL TMB reagent per well.

Develop for 10 - 30 min.

Quench using 30 µL 10% phosphoric acid per well.

Read plate on the Spectramax or similar plate reader. 384-495 nm for unquenched reactions, 450 nm for quenched reactions.